U.S. patent application number 11/169833 was filed with the patent office on 2005-10-27 for identification of genes having a role in the presentation of diabetic nephropathy.
Invention is credited to Brady, Hugh Redmond, Godson, Catherine Mary, Martin, Finian Mary, McMahon, Ruth Anne, Murphy, Madeline Anne.
Application Number | 20050239129 11/169833 |
Document ID | / |
Family ID | 11042009 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050239129 |
Kind Code |
A1 |
Brady, Hugh Redmond ; et
al. |
October 27, 2005 |
Identification of genes having a role in the presentation of
diabetic nephropathy
Abstract
A method for identifying a gene having a role in the
presentation of diabetic nephropathy comprises culturing mesangial
cells in the presence of a concentration of glucose sufficient to
induce differential expression, especially up-regulation, of a gene
susceptible to such differential expression and identifying the
gene so induced. The cells are also optionally subjected to
mechanical strain and/or TGF-.beta.1 can be added to the culture
medium. The differentially expressed genes can be identified by
suppression subtractive hybridisation. The method has resulted in
the identification of novel genes which play a role in the
presentation of diabetic nephropathy. The genes can be used as
diagnostic markers for diabetic nephropathy and as the basis of
drug development programmes.
Inventors: |
Brady, Hugh Redmond;
(Dublin, IE) ; Godson, Catherine Mary; (Dublin,
IE) ; Martin, Finian Mary; (Dublin, IE) ;
McMahon, Ruth Anne; (Dublin, IE) ; Murphy, Madeline
Anne; (Dublin, IE) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
11042009 |
Appl. No.: |
11/169833 |
Filed: |
June 30, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11169833 |
Jun 30, 2005 |
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09914191 |
Aug 24, 2001 |
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09914191 |
Aug 24, 2001 |
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PCT/IE00/00026 |
Feb 28, 2000 |
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Current U.S.
Class: |
435/6.13 ;
435/455 |
Current CPC
Class: |
C12Q 1/6809
20130101 |
Class at
Publication: |
435/006 ;
435/455 |
International
Class: |
C12Q 001/68; C12N
015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 26, 1999 |
IE |
990157 |
Claims
1. A method of diagnosing and determining the progression of
diabetic nephropathy which comprises, culturing mesangial cells in
a medium in the presence of transforming growth factor .beta.1
(TGF-.beta.1) and a concentration of glucose sufficient to induce
differential expression of a gene susceptible to such differential
expression; identifying the gene so induced by suppression
subtractive hybridization; and using said gene as a diagnostic
marker for the progression and presentation of diabetic
nephropathy.
2. A method of identifying drugs for use in the prevention and/or
therapy of diabetic nephropathy which comprises, culturing
mesangial cells in a medium in the presence of transforming growth
factor .beta.1 (TGF-.beta.1) and a concentration of glucose
sufficient to induce differential expression of a gene susceptible
to such differential expression; identifying the gene so induced by
suppression subtractive hybridization; and screening potential drug
compounds for an effect on the identified gene or on a protein
encoded by said gene.
3. The method according to claim 1, wherein the mesangial cells are
cultured in the presence of a concentration of glucose sufficient
to induce up-regulation of a gene susceptible to such
up-regulation.
4. A method according to claim 1, wherein the concentration of
glucose is greater than 5 mM.
5. A method according to claim 1, wherein the mesangial cells are
subjected to mechanical strain.
6. A method according to claim 1, wherein transforming growth
factor .beta.1 (TGF-.beta.1) is added to the culture medium.
7. A method according to claim 1, wherein the possibility of
differential expression due to hyperosmolarity is excluded.
8. A method according to claim 1, wherein the gene so
differentially expressed is SEQ ID NO: 1 or SEQ ID NO:3.
9. The method according to claim 2, wherein the mesangial cells are
cultured in the presence of a concentration of glucose sufficient
to induce up-regulation of a gene susceptible to such
up-regulation.
10. A method according to claim 2, wherein the concentration of
glucose is greater than 5 mM.
11. A method according to claim 2, wherein the mesangial cells are
subjected to mechanical strain.
12. A method according to claim 2, wherein transforming growth
factor .beta.1 (TGF-.beta.1) is added to the culture medium.
13. A method according to claim 2, wherein the possibility of
differential expression due to hyperosmolarity is excluded.
14. A method according to claim 2, wherein the gene so
differentially expressed is SEQ ID NO: 1 or SEQ ID NO:3.
Description
TECHNICAL FIELD
[0001] This invention relates to the characterisation and
identification of genes which play a role in diabetes, more
particularly in the onset and progression of diabetic nephropathy
and to the use of genes so characterised and/or identified as
diagnostic markers for diabetic nephropathy and as the basis of
drug development programmes.
BACKGROUND ART
[0002] Between 2-5% of the population develops diabetes mellitus
and 20-30% of diabetics develop diabetic nephropathy. The latter
accounts for over 30% of end-stage renal failure (E.S.R.F.)
requiring dialysis or transplantation in western society. The
pathological hallmark of diabetic nephropathy is glomerulosclerosis
due to accumulation of extracellular matrix proteins in the
glomerular mesangium. Mesangial matrix accumulation reflects both
increased synthesis and decreased degradation of extracellular
matrix (ECM) components, and correlates with the clinical onset of
proteinuria, hypertension and progressive kidney failure.
Hyperglycaemia is a major stimulus for mesangial cell matrix
production in diabetic nephropathy. The mechanisms by which
hyperglycaemia perturb mesangial cell function are still being
appreciated and include direct effects of high extracellular
glucose levels and indirect effects transduced through alterations
in glomerular haemodynamics and through the actions of advanced
glycosylation end products.
[0003] Propagation of mesangial cells under conditions of high
ambient glucose has proved a useful in vitro model with which to
probe the molecular basis for mesangial matrix accumulation in
diabetes, attributable to hyperglycaemia. Specifically, exposure of
cultured mesangial cells to high glucose stimulates de novo
synthesis of ECM components, such as type IV collagen, fibronectin
and laminin, and other products that are accumulated in vivo (Ayo,
S. H., et al. (1990) Am. J. Pathol. 136, 1339-1348; Wahab, N. A.,
et al. (1996) Biochem. J. 316, 985-992; and Ayo, S. H., et al.
(1991) Am. J. Physiol. 260, F185-F191).
[0004] In view of the high morbidity and mortality rate from
diabetic nephropathy in diabetics there is a need to identify
stimuli which affect the onset and progression of diabetic
nephropathy with the aim of preventing such onset or inhibiting or
limiting the progression thereof.
DISCLOSURE OF INVENTION
[0005] The invention provides a method for identifying a gene
having a role in the presentation of diabetic nephropathy, which
method comprises culturing mesangial cells in a medium in the
presence of a concentration of glucose sufficient to induce
differential expression of a gene susceptible to such differential
expression and identifying the gene so induced by suppression
subtractive hybridisation.
[0006] Preferably, the mesangial cells are cultured in the presence
of a concentration of glucose sufficient to induce up-regulation of
a gene susceptible to such up-regulation.
[0007] Further, preferably, the concentration of glucose is greater
than 5 mM.
[0008] A concentration of 5 nM falls within the normal range of
plasma glucose levels in a healthy human subject (4.2-6.4
mmol/l).
[0009] The concentration of glucose used is suitably in the range
5-30 mM. The concentration of 30 mM was chosen as the classic "in
vitro" model of diabetic nephropathy which induces changes in
mesangial function that mimic human disease. This level is also
encountered in many diabetics in vivo.
[0010] In one embodiment, the mesangial cells are subjected to
mechanical strain.
[0011] In a further embodiment, transforming growth factor .beta.1
(TGF-.beta.1) is added to the culture medium.
[0012] Suppression subtractive hybridisation (SSH) is a method
based on suppressive PCR that allows creation of subtracted cDNA
libraries for the identification of genes differentially expressed
in response to an experimental stimulus (Gurskaya, N. G., et al.
(1996) Anal. Biochem. 240, 90-97). SSH differs from earlier
subtractive methods by including a normalisation step that
equalises for relative abundance of cDNAs within a target
population. This modification should enhance the probability of
identifying increased expression of low abundance transcripts, and
represents a potential advantage over other methods for identifying
differentially regulated genes such as differential display-PCR
(DD-PCR) (Liang, P., and Pardee, A. B. (1992) Science 257, 967-97)
and cDNA-representation difference analysis (Hubank, M., and
Schatz, D. G., (1994) Nucleic Acid Res. 22, 5640-5648).
[0013] To date we have used SSH to identify 150 genes
differentially induced when human mesangial cells were exposed to
high extracellular glucose (defined herein as 30 mM versus 5 mM) in
vitro. These genes included:
[0014] (a) known regulators of mesangial cell activation in
diabetic nephropathy, namely fibronectin, caldesmon, thrombospondin
and plasminogen activator inhibitor-1;
[0015] (b) novel genes; and
[0016] (c) genes whose induction by high glucose has not previously
been reported as hereinafter described.
[0017] Prominent among the latter were genes encoding
cytoskeleton-associated proteins and connective tissue growth
factor (CTGF), a modulator of fibroblast matrix production. We have
also demonstrated elevated CTGF mRNA levels in glomeruli of rats
with streptozotocin-induced diabetic nephropathy.
[0018] In one aspect of the invention, the possibility of
differential expression due to hyperosmolarity is excluded.
[0019] Hyperosmolarity is, however, a component of diabetic
nephropathy and thus hyperosmolarity may represent a mechanism
through which high glucose induces differential expression of
certain genes having a role in the presentation of the disease.
[0020] For example, we have shown that mannitol provoked less
mesangial cell CTGF expression in vitro than high glucose,
excluding hyperosmolarity as the key stimulus.
[0021] High glucose also stimulated expression of transforming
growth factor .beta.1 (TGF-.beta.1) and addition of TGF-.beta.1 to
mesangial cells triggered CTGF expression. Anti-TGF-.beta.1
antibody blunted CTGF expression induced by high glucose. Together,
these data suggest that (1) high glucose stimulates mesangial CTGF
expression by TGF.beta.1-dependent and independent pathways, and
(2) CTGF may be a mediator of TGF-.beta.1-driven matrix production
within a diabetic milieu.
[0022] CTGF may therefore be an attractive target for design of
novel anti-sclerotic therapies for diabetic glomerulosclerosis.
[0023] CTGF derived from mesangial cells is a potential stimulus
for increased synthesis of ECM proteins and mesangial expansion in
diabetic nephropathy. The mechanisms by which high glucose triggers
mesangial cell CTGF and, indeed, TGF-.beta., mRNA expression remain
to be defined. Possible upstream triggers of CTGF transcription in
response to high glucose include de novo synthesis of diacylglcerol
(DAG) and subsequent activation of protein kinase C (PKC)
(DeRubertis, F. R., and Craven, P., (1994) Diabetes 43, 1-8 and
Fumo P., et al. (1994) Am. J. Physiol. 267, F632-F638),
non-enzymatic glycation end-products (Brownlee, M., et al. (1984)
Ann. Intern. Med 101, 527-537 and Cohen, M. P., and Ziyadeh, F. N.
(1994) Kidney Int. 45, 475-484) increased activity of the polyol
pathway and disordered myoinositol metabolism (Goldfarb, S., et al.
(1991) Diabetes 40, 465-471 1991) or through the recruitment of
locally generated growth factors such as TGF-.beta.1 and other
mediators. (Sharma K., and Ziyadeh, F. N., (1995) Diabetes 44,
1139-1146).
[0024] TGF-.beta.1 has been implicated as the key mediator of
extracellular matrix accumulation in diabetic nephropathy and other
chronic renal disease. Several studies have reported increased
expression of TGF-.beta.1 in renal glomeruli in human and
experimental models of diabetes (Park, I., et al. (1997) Diabetes
46, 473-480; Sharma, K., (1996) Diabetes 45, 522-530). Short term
administration of TGF-.beta.1 neutralising antibodies attenuates
overexpression of mRNAs encoding matrix components and
glomerulosclerosis in the STZ mouse model of diabetes (Park, I., et
al. (1997) Diabetes 46, 473-480). CTGF shares some of the
biological actions of TGF-.beta.1 such as stimulation of cell
proliferation and extracellular matrix protein synthesis in
fibroblasts. When considered in this context, the results described
herein suggest that TGF-.beta.1 may promote mesangial matrix
production, in part, by inducing CTGF synthesis. TGF-.beta.1 has a
complex profile of biological activities that includes
pro-inflammatory, pro-fibrotic and anti-inflammatory effects. By
targeting CTGF it may be possible to attenuate the
sclerosis-inducing effects of TGF-.beta.1 while preserving its more
desirable anti-inflammatory activities.
[0025] Novel genes identified by the method according to the
invention are identified herein as IHG (Increased in High Glucose)
and DHG (Down in High Glucose) and are represented by genes which
include the following sequences 1, 3 and 4:
1 (SEQ ID NO:1) 1)TTGGAATAGTTCTTGCTTTATAAAAATAGTACTGCGATTAA- A
AAAAAAGCACTTCTGCCAAAGGAACCATGTTCCAACACCGCA
AACAAGGTGTTCTGCTTAAACAGAGTAAGATACACCACCCCC ATCCATCCCTTCCTTCCCTGTTC-
CCCTCCCAACTTGAGTTGTGT CATTCGCACCAGTGTCCTGGGTGGTAGGGATGCTACAGCCAC
CTAAGGCAAGGAGCCCTGGGAGGTGGGAGGGCTTGCATGGTT
AAGCACACCAGAACTGAAGCGCAAAAGGGTCAGCTGTCTTCA TCTAGAATCTCTGGATGTTCCTT-
CCAGAAAGCATCCCCGATGA TATCGCAGTGCAAGGGCACTGGCTTTGTCCTGGTCCGGGTCAC
TGCCATCTTTTTTCCTTCCATTTCTGTTGGCAGCTTAATTTCTTT
TGTCATCACTTCATCCACCTTCTGCCATATCAACACAGTCCCTT
TCCTATACATCGGCAGCTCATTATTATAGTTGATGTTGAATTC
AGAAAACAAAATCTCATTCTTGTCTGCTGNAAGAGTTCCCTGT
AATCTCCCTTGGGCTTGTACTGGTGTTAGTCCAGATTGTTG (SEQ ID NO:2)
...........................................
.................................GGTCCTTTAA
AGTCTGGTTGCTGGGATACACCACGACTCTTCCGGTCAAAGCC
TGGGGGATACAGAAGGGGCTRGTCCTCAAAGTAATCCCGCCA ATAAAACAYATAGCTGGAGGCAA-
ACTGGGAGGYCACGTGAGT CATGAACTTTACTGGCTCTTCTTTTAAACCAATTGGTTTTCCGC
TTGWACACAAAGCTGTACTCATCACTCTGTCCATAACGCGAT
CACAATATCCTCTAGTTCTTCCATCACAGTCTGCGCACATTTG
GTCATCAGCTGGAGAGCACGGCTGTCATTGGGTTTTGCAAAGT
TGTGCTTCTCAGCAAACCGATGGAAATTCCGGCCGTCCAGCCG
NACTACCACCCAGCAGTGTGCCAGGCAGGTGTCGTCAGCCTC GAAGTCCCTCACGTACTCGAACT-
TGCTTTTTGCCATGGTCGCC CCCAATCTCAGGTACCGTCTCAGAGTGATGGAAATGGTGGCC
AAGGAATCGTGAACCTTAACCTTTACAGGCGCCCCACATTCTAC
ACGCGGAAAGGAAAGGGCCAGATAGCCCCGCCCCGGAAGTG TTCTCTTCGTGGCTACTCTAGCCG-
TAGGGCGGTCATAGTCTCT CTCGSCTCTCCCTGKAGTTCTTAAMCYYCCAGGGAAARAGGA
TGGAGGTTTAGGTTCCTCCGTTAGCACCTTCCACGCTTGCTTCT
TCCTCCTCCCGGTCTGCGGCAAATCAGTCTCACGAGGTTTTTA
AAAATTATTTTTTATCTGCTGGCCTT (SEQ ID NO:3)
............................ATGACACAAATATTAG
GATTTTTATTTTTACTATTATCCACCAGCAACAAGATATCAAAC
ACTGGTTCTGTGATTATTTAATGGTGAAAAAGTTGAATAAATC
AATTTAGTATACCCATATGTTGGAATATTGAGTCCATTTTTCTT
TTAAAAATCACACTTTGGAATAATTGATGATACTGGCAAATGC
TCAAGCTGAGTGGAAAAATATATAAACATTGTATAGGCGAAT AATTCCAATCTTGTGCATTCCCT-
GTGTAAACCTACATACACAA AAAGAAAAAAGACTGAAAGGAACCATCCACAATGCTTTGATC
GGGAAAGACGGAGAAACAAAGTGTTAATTTTCTTAACTATAG
TTTTNGGTGTATTCCAGATTTTCTACAAGTTAATA(IHG-1); (SEQ ID NO:4)
2)GTACTTTGGATTTGGTTAACCTGTTTTCTTCAAGCCTGAGGT
TTTATATACAAACTCCCTGAATACTCTTTTTGCCTTGTATCTTC
TCAGCCTCCTAGCCAAGTCCTATGTAATATGGAAAACAAACA CTGCAGACTTGAGATTCAGTTGC-
CGATCAAGGCTCTGGCATTC AGAGAACCCTTGCAACTCGAGAAGCTGTTTTTATTTCGTTTTT
GTTTTGATCCAGTGCTCTCCCATCTAACAACTAAACAGGAGCC
ATTTCAAGGCGGGAGATATTTTAAACACCCAAAATGGTTGGG TCTGATTTTCAAACTTTTAAAAC-
TTCACTACTGATGATTCTGCA CGCTAAGGCGAATTTGGTCCAAACACATAAGTGTGTGTGTTTT
GTATACACTGTATGACCCCACCCCAAATCTTTGTATTGTCCAC ATTCTCC IHG-2; (SEQ ID
NO:5) 3)AGAAGCCAATTTAGGAANCCNACAGNAAANAAATGC- TGTTTT
ATAGCAGAGAAAACACGGCACACCAAGGTTAAGTAGTTTGTA
GACGATGTTGAATAGGTTCAGGTACAGGTCAATGCAGTGATG AGGAAAGCACCTANGTATACTTG-
ACAGATAGTCCCCTTTGCTT AACACCCAACTCCTCCACCCTGTGCAGTTTNNCTTGTGCCAGT
GATCACAGGATTCGCTGAGTGAATTACCATAATTGGATTTAT
TCACGAAGGGGATGTTTTC(IHG-3); and (SEQ ID NO:6)
4)ATTGATAGAGGCCCTGTFFCATGACATTTCATGAGTTTCAAT
ATGTTGTTCAGCATGTTGTGAGGTGACTCTCAGCCCCTTTCCC
ACTGAGATGGACTGTGGTGATGCTGTGAGGCTGTGACTGACA CACCTTCATGTGCCCAAGCATGG-
GTTTGATCACAGGTCACATG CAGTTTTTGGCATAGTAAATGTATCATTGTTCTTTTCCTCCCTC
CTAAAGGAAACAGAGGAATCCACCTGTATGAGAGTGCCATGT
AGGGATAAACTTAAGGACAGATGACACATTGGTCATGTTCG TGATAAGGAAA(DHG-1).
[0026] The invention also provides SEQ ID NOS: 1-3, 5 and 6 set out
above.
[0027] In initial studies the gene IHG-2 was assumed to be new.
However, as hereinafter demonstrated IHG-2 was identified as being
a formerly unknown part of the gremlin gene. We have found that
mesangial cell gremlin mRNA levels are induced by high glucose,
cyclic mechanical strain and TGF-.beta.1 in vitro, and gremlin mRNA
levels are elevated in the renal cortex of rats with
streptozotocin-induced diabetic nephropathy in vivo. Gremlin
expression was observed in parallel with induction of bone
morphogenetic protein-2 (BMP-2), a target for gremlin in models of
cell differentiation.
[0028] Gremlin, together with DAN and cerberus, are members of the
cysteine knot super-family of proteins that have recently been
shown to play important roles in limb development and neural crest
cell differentiation (Hsu, D. R., et al (1998) Mol. Cell. 1,
673-83; Zuniga, A., et al. (1999) Nature 401, 598-602). Of
potential interest in the context of diabetic nephropathy, gremlin
is a putative inhibitor of BMP-2 in models of neural crest cell
differentiation. BMP-2 has recently been reported to have
antiproliferative effects on mesangial cells.
[0029] The following Examples show that (a) IHG-2 is part of
gremlin, (b) gremlin is expressed in diabetic nephropathy in vivo,
(c) both glycemic and mechanical strain stimulate mesangial cell
gremlin expression in vitro, (d) high glucose induces gremlin, in
part, through TGF.beta.-mediated pathways, and (e) gremlin is a
potential endogenous antagonist of BMPs within a diabetic
glomerular milieu.
[0030] The invention also provides use of a gene identified by the
method according to the invention:
[0031] 1) as a diagnostic marker for the progression and
presentation of diabetic nephropathy;
[0032] 2) as an index of disease activity and the rate of
progression of diabetic nephropathy; and
[0033] 3) as a basis for identifying drugs for use in the
prevention and/or therapy of diabetic nephropathy.
[0034] Thus, it will be appreciated that early diagnosis of
diabetic nephropathy based on diagnostic markers identified in
accordance with the invention can be used in conjunction with
aggressive therapies to prevent full blown development of diabetic
nephropathy.
[0035] The level of expression of genes identified in accordance
with the invention could correlate with the degree of disease
progression.
[0036] Furthermore, genes identified in accordance with the
invention can represent novel therapeutic targets for drug
development programmes. Once it has been established that a given
gene has a designated role in the pathophysiology of diabetic
nephropathy, the development of new therapeutic agents (such as,
for example, small molecules, recombinant inhibitors and receptor
antagonists) could be designed to inhibit expression of these genes
and, thereby, prevent the development of diabetic nephropathy.
[0037] Genes identified in accordance with the invention can also
be used as a clinical index of progressive renal sclerosis and
scarring, as a guide to the response of progressive diabetic
nephropathy to therapy and also as markers of the prevention or
development thereof.
[0038] It is possible to generate mouse knock-out (k/o) models for
genes identified in accordance with the invention and to generate
diabetic k/o mouse models, (for example by treatment with
streptozotocin) and determine if onset of diabetic nephropathy is
inhibited, reduced or delayed. Thus one can determine if a given
knock-out gene has a definite role in the progression and
development of diabetic nephropathy.
BRIEF DESCRIPTION OF DRAWINGS
[0039] FIG. 1 is an autoradiograph of CTGF levels analysed by
Northern Blot as described in Example 2;
[0040] FIG. 2 is a graph of the relative amount of CTGF mRNA as
estimated by Phosphor Imager quantification as described in Example
2;
[0041] FIG. 3 is a 2% agarose gel showing ethidium-stained PCR
products as described in Example 2;
[0042] FIG. 4 is a nucleotide sequence alignment of the rat CTGF
transcript and the mouse CTGF homologue fisp 12 as described in
Example 3;
[0043] FIG. 5 is an amino acid sequence alignment of the rat CTGF
transcript and the mouse CTGF homologue fisp 12 as described in
Example 3;
[0044] FIG. 6 is a 2% agarose gel showing ethidium-stained PCR
products as described in Example 3;
[0045] FIG. 7 is an autoradiograph of CTGF levels in the presence
of TGF-.beta.1 and TGF-.beta.1 neutralising antibodies analysed by
Northern Blot as described in Example 4;
[0046] FIG. 8 is a graph of the relative amount of CTGF mRNA as
estimated by Phosphor Imager quantification as described in Example
4;
[0047] FIG. 9 is an autoradiograph of CTGF levels in the presence
of varying amounts of glucose and TGF-.beta.1 neutralising
antibodies analysed by Northern Blot as described in Example 4;
[0048] FIG. 10 is a graph of the relative amount of CTGF mRNA as
estimated by Phosphor Imager quantification as described in Example
4;
[0049] FIG. 11 is an autoradiograph of CTGF levels in the presence
of varying amounts of glucose and PKC inhibitor GF102903X analysed
by Northern Blot as described in Example 4;
[0050] FIG. 12 is a graph of the relative amount of CTGF mRNA as
estimated by Phosphor Imager quantification as described in Example
4;
[0051] FIG. 13 is a graphical representation of a BLAST output from
the EST database as described in Example 6;
[0052] FIG. 14 is a graphical representation of the alignment of
human gremlin and rat drm when compared using the BLAST algorithm
as described in Example 6;
[0053] FIG. 15 is the sequence of mesangial cell gremlin cDNA;
[0054] FIG. 16 is an autoradiograph of IHG-2, gremlin, fibronectin
and GAPDH mRNA levels analysed by Northern Blot as described in
Example 7;
[0055] FIG. 17 is a graph of relative mRNA levels as estimated by
Phosphor Imager quantification as described in Example 7;
[0056] FIG. 18 is an autoradiograph of gremlin, fibronectin and
GAPDH mRNA analysed by Northern Blot as described in Example 7;
[0057] FIG. 19 is a further graph of relative mRNA levels as
estimated by Phosphor Imager quantification as described in Example
7;
[0058] FIG. 20 is an autoradiograph of gremlin mRNA levels in the
kidney cortex of a STZ-diabetic rat and an age matched control
analysed by Northern Blot as described in Example 7;
[0059] FIG. 21 is a further graph of relative mRNA levels as
estimated by Phosphor Imager quantification as described in Example
7;
[0060] FIG. 22 is an autoradiograph of gremlin, fibronectin and
GAPDH mRNA levels analysed by Northern Blot as described in Example
8;
[0061] FIG. 23 is a graph of relative mRNA levels as estimated by
Phosphor Imager quantification; and
[0062] FIG. 24 is a graphical representation of representative
reactions of four independent experiments as described in Example
9.
[0063] The invention will be further illustrated by the following
Examples:
MODES FOR CARRYING OUT THE INVENTION
EXAMPLE 1
Identification of Mesangial Cell Genes Differentially Induced by
High Glucose
[0064] a) Cell Culture and Streptozotocin-Induced Diabetic Rats
[0065] Primary human mesangial cells were cultured as previously
reported (Brady, H. R., et al. (1992) Kidney nt. 42, 480-487 and
Denton, M. D., et al. (1991) Am. J. Physiol, 261, F1071-F1079).
Cells (passage 7-11) were maintained in medLum (Clonetics)
containing either 5 mM or 30 mM D-glucose for 7 days. Culture
medium was replenished three times during this period to maintain
glucose levels in the desired range. To control for the effects of
hyperosmolarity, mesangial cells were cultured in media containing
5 mM glucose supplemented with 25 mM mannitol.
[0066] Male Munich-Wistar rats (260-290 g, Simonsen Laboratories)
were rendered diabetic by treatment with streptozotocin (STZ;
Sigma), 50 g/kg, intravenously as described previously (Zatz, R.,
et al. (1985). Proc. Natl. Acad. Sci. USA. 82, 5963-5967). At
months 2 and 4 after induction of diabetic nephropathy (DN), rats
were anaesthetized with intraperitoneal injection of pentobarbital
(50 mg/kg), and the right kidney was excised and weighed
immediately. Glomeruli were isolated from renal cortex by the
standard sieving method (Brady, H. R. et al. (1992) and Denton, M.
D. et al. (1991) supra). Glomerular isolation was completed within
20 minutes of removing the kidney. RNA extraction proceeded
immediately thereafter.
[0067] b) RNA Isolation
[0068] Polyadenlyated RNA was isolated from mesangial cells using
the Microfast Track (Microfast Track is a Trade Mark) kit
(Invitrogen). Total RNA was isolated from glomeruli using RNAzol
solution (TEL-test Inc.).
[0069] c) Suppression Subtractive Hybridisation (SSH)
[0070] SSH was performed with the PCR-SELECT cDNA subtraction kit
(Clontech) as directed by the manufacturer with the modification
that a four-fold greater than recommended amount of driver cDNA was
added to the second hybridisation. Starting material consisted of 2
.mu.g of mesangial cell mRNA cultured in 30 mM D-glucose for 7 days
as "tester" and 2 .mu.g of mesangial cell mRNA cultured in 5 mM
D-glucose for 7 days as "driver". Thirty primary PCR cycles and 12
secondary PCR cycles were performed.
[0071] d) Cloning and Sequencing of cDNAs
[0072] PCR products generated by SSH were subcloned into the PCR
2.1 vector using the original TA cloning kit (Invitrogen).
Subcloned cDNAs were isolated by colony PCR amplification.
Sequencing was performed using an automated ABI 370A DNA sequencing
system. Sequence reactions were carried out with the ABI prism dye
terminator cycle sequencing ready reaction kit (Perkin Elmer). The
sequences obtained were compared against GenBank/EMBL and Expressed
Sequence Tag (EST) databases using BLAST searches.
[0073] SSH analysis suggested differential induction of 16 mRNAs in
primary cultures of human mesangial cells propagated for 7 days in
30 mM glucose. Northern Blots performed using formaldehyde
denaturation according to standard protocols and quantitated using
a Phosphor Imager (Biorad) confirmed differential expression of
fifteen of the sixteen subcloned fragments as indicated in Table 1.
In Table 1.sup.a refers to the sequence identity based on
comparisons with the Genbank/EMBL database;
[0074] .sup.b refers to an estimate of the size (kb) of the mRNA
identified by Northern Blot analysis; and
[0075] .sup.c refers to the differential expression of each gene
based on Northern Blot analysis of primary human mesangial cells
cultured under indicated conditions relative to expression in cells
cultured in 5 mM glucose. Values were obtained by Phosphor-Imaging
and were normalised by comparison with glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (*, detected in mesangial cells cultured in 5
mM glucose+25 mM mannitol and 30 mM glucose, but not in 5 mM
glucose, fold expression is degree of expression relative to that
found in 5 mM glucose).
2TABLE 1 Summary of cDNAs identified by SSH as being induced in
mesangial cells cultured in high glucose. Differential
Expression.sup.c 25 mM 30 mM mannitol + 5 mM Gene.sup.a
mRNAkb.sup.b glucose glucose Extracellular Matrix Proteins
Fibronectin 7.0 2.1-fold 1.5-fold Thrombospondin 6.0 7.0-fold
8.0-fold Actin-Binding Proteins MRLC 0.9 3.9-fold 1.6-fold
T-plastin 1.2 4.2-fold 1.8-fold Caldesmon 3.6 3.3-fold 2.8-fold
Profilin 1.0 2.2-fold 2.3-fold CAP 2.6 1.5-fold 1.7-fold ARP3 2.5
2.0-fold 1.0-fold Growth Factors CTGF 2.4 3.0-fold 1.5-fold Others
PAI-1 2.0 1.2-fold 1.0-fold 3.0 3.9-fold 2.0-fold RBM3 1.5 1.8-fold
1.4-fold Ubiquitin 3.0 2.3-fold 1.7-fold TCTP 0.8 4.3-fold 2.5-fold
IHG-1 3.4 * * IHG-2 2.5 2.0-fold 2.0-fold
[0076] Sequence analysis revealed induction of four genes
implicated previously in the pathogenesis of diabetic nephropathy:
fibronectin, caldesmon, PAI-1, and thrombospondin. Of eleven other
cDNA fragments, one encoded a novel gene, designated herein as
IHG-1 and ten encoded known genes, including IHG-2 as hereinafter
described, whose induction by high glucose had not been reported
previously. Prominent among the latter genes were connective tissue
growth factor (CTGF) and several cytoskeleton-associated proteins,
namely profilin, caldesmon, adenyl cyclase-associated protein
(CAP), actin-related protein-3 (ARP3), T-plastin, and myosin
regulatory light chain (MRLC). Subsequent studies focused on
induction of CTGF, a regulator of matrix production in several
model systems as described in Examples 2 and 3. The prominence of
genes encoding multiple actin-binding proteins is also noteworthy,
given recent reports implicating F-actin disassembly in the
pathogenesis of mesangial cell dysfunction and glomerular
hypertension in diabetic nephropathy (Zhou, X., et al. (1995) Lab.
Invest. 73, 372-383 and Zhou, X. Lai, et al. (1997) Kidney Int. 51,
1797-1808). The induction of profilin expression is particularly
interesting given its role as a regulator of actin polymerization
under conditions of cell stress (Sohn, R. H. and
Goldschmidt-Clermont, P. J. (1994) Bioessays 16, 465-472).
[0077] Within a diabetic milieu, high glucose levels may perturb
cellular function through glucose-specific actions or by increasing
the osmolarity of extracellular fluids. The role of hyperosmolarity
as a mediator of gene induction by high glucose was assessed by
comparing mRNA levels, as determined by Northern Blot, in cells
cultured in either 30 mM glucose or in 5 mM glucose supplemented
with 25 mM mannitol. High glucose was more effective than high
osmolarity at inducing expression of CTGF, myosin regulatory light
chain (MRLC), actin related protein 3 (ARP3), T-plastin and
translationally controlled tumor protein (TCTP). High glucose and
mannitol-induced hyperosmolarity afforded equivalent induction of
the other products.
EXAMPLE 2
CTGF Expression in Mesangial Cells Cultured in High Glucose
[0078] a) Influence of High Ambient Glucose on CTGF mRNA Levels in
Human Mesangial Cells.
[0079] CTGF, is a 38 kD cysteine-rich secreted peptide known to
modulate ECM production in some extrarenal cell types. In Example
1, SSH analysis identified a cDNA fragment of 250 bp which was
identical to bases 814-1061 of the human CTGF cDNA. Induction of
CTGF mRNA expression in primary human mesangial cells cultured in
high glucose was investigated by Northern Blotting as shown in FIG.
1.
[0080] In FIG. 1 the lanes represent the following:
[0081] Lane 1: RNA from mesangial cells exposed to 5 mM
glucose;
[0082] Lane 2: RNA from mesangial cells exposed to 5 mM glucose and
25 mM mannitol;
[0083] Lane 3: RNA from mesangial cells exposed to 30 mM glucose
for seven days.
[0084] A 2.4 kb band was detected following hybridisation with the
CTGF probe. The relative amounts of CTGF mRNA as estimated by
Phosphor Imager quantification are indicated in FIG. 2. All of the
values were normalised to GAPDH levels and the results are
representative of three independent experiments.
[0085] The results indicate that CTGF mRNA expression was between
2.5-3.3-fold higher in mesangial cells cultured in 30 mM glucose as
compared with 5 mM glucose.
[0086] b) Effect of CTGF on Mesangial Cell Matrix Production.
[0087] To investigate the direct effects of CTGF up-regulation on
matrix production, in particular the effect on collagens I and IV
and fibronectin, mesangial cells were incubated with recombinant
CTGF protein.
[0088] Mesangial cells were serum starved for 24 hr in RPMI 1640
medium supplemented with 0.5% fetal bovine serum (FBS) and then
exposed to rhCTGF (8 ng/ml) (a generous gift from Dr. Gary
Grotendorst) for 24 hr (Kreisberg, J. I. and Ayo, S. H. (1993).
Kidney Int. 43, 109-113). Total RNA was extracted and chromosomal
DNA was removed using DNase 1 (Gibco-BRL). Equal amounts of cDNA
were subsequently amplified by PCR using specific primers for GAPDH
(Gen/EMBL accession no. AJ005371, sense: ACCACAGTCCATGCCATCAC (SEQ
ID NO: 7); antisense: TCCACCACCCTGTTGCTGTA (SEQ ID NO: 8), Collagen
I (Gen/EMBL accession no. X55525, sense: GGTCTTCCTGGCTTAAAGGG (SEQ
ID NO: 9); antisense: GCTGGTCAGCCCTGTAGAAG (SEQ ID NO: 10)),
Collagen IV (Gen/EMBL accession no. M11315, sense:
CCAGGAGTTCCAGGATTTCA (SEQ ID NO: 11); antisense:
TTTTGGTCCCAGAAGGACAC (SEQ ID NO: 12) and fibronectin (Gen/EMBL
accession no. X02761, sense: CGAAATCACAGCCAGTAG (SEQ ID NO. 13),
antisense: ATCACATCCACACGGTAG (SEQ ID NO: 14)).
[0089] FIG. 3 depicts ethidium-stained panels of a 2% (w/v) agarose
gel containing 10 .mu.l of each PCR reaction after
electrophoresis.
[0090] In FIG. 3 the lanes represent the following:
[0091] Lane 1: RT-PCR products from mesangial cells cultured in
RPMI 1640 and 0.5% FBS;
[0092] Lane 2: RT-PCR products from mesangial cells exposed to
rhCTGF (8 ng/ml) for 24 hr.
[0093] These results indicate that rhCTGF up-regulates mesangial
cell collagens I and IV and fibronectin. These proteins typify
matrix accumulation as seen in diabetic nephropathy.
[0094] CTGF is a member of a small family of highly homologous
proteins termed the CCN family (for CTGF/fisp-12, ceflo/cyr61 and
Nov) (Bork, P (1993). FEBS Letts. 327, 125-130.). These peptides
are characterised by conservation of 38 cysteine residues that
constitute more than 10% of the amino acid content. All members
have signal peptides and appear to be secreted via orthodox
secretory pathways (Bradham, D. M., (1991) J. Cell. Biol. 114,
1285-1294). In the context of diabetic nephropathy, it is
intriguing that CTGF which is up-regulated in the presence of
ambient glucose, in turn, up-regulates the production of
extracellular matrix (ECM). These data demonstrate the potential of
CTGF as a stimulus for increased ECM synthesis and mesangial
expansion in diabetic nephropathy.
EXAMPLE 3
Enhanced CTGF Expression in Renal Cortex and Isolated Glomeruli of
Rats with STZ-Induced Diabetic Nephropathy
[0095] To assess CTGF expression in diabetic nephropathy in vivo,
CTGF mRNA levels were measured in RNA isolated from the cortex of
rats with STZ-induced diabetes mellitus. To this end, PCR primers
for rat CTGF were designed from the sequence of the mouse CTGF
homologue, fispl2 (Genbank/EMBL accession no. M70642, sense:
CTAAGACCTGTGGAATGGGC (SEQ ID NO: 15); antisense:
CTCAAAGATGTCATTGTCCCC (SEQ ID NO: 16)) (Ryseck, R. P., (1991) Cell
Growth Differ. 2, 225-233).
[0096] RT-PCR was performed on total RNA extracted from renal
cortex of STZ-diabetic rats and age matched controls. The sequence
of the rat CTGF transcript was 94% identical at the nucleotide
level (FIG. 4) and 99% identical at the amino acid level (FIG. 5)
to the mouse CTGF homologue fispl2 (bases 783-1123, accession no.
M70642). Nucleotides that differ between the two species are given
in upper case and the single different amino acid is in bold.
[0097] Induction of CTGF mRNA was observed in the renal cortex of
rats with STZ-induced diabetic nephropathy at four months after
administration of STZ, coincident with mesangial expansion and
proteinuria as shown in FIG. 6 and data not shown.
[0098] FIG. 6 depicts ethidium-stained panels of a 2% (w/v) agarose
gel containing 10 .mu.l of each PCR reaction after electrophoresis.
CTGF and GAPDH mRNA levels were analysed in total RNA purified from
2 diabetic animals with established nephropathy after four months
of diabetes (lanes 1 and 2) and two age matched control animals
(lanes 3 and 4).
[0099] CTGF expression was further localized to glomeruli by RT-PCR
analysis of RNA extracted from glomeruli isolated by differential
sieving from the renal cortex of rats with STZ-induced diabetic
nephropathy. Glomerular levels of CTGF mRNA were increased by
2.5-fold and 1.6-fold after two months and four months of diabetes,
respectively, by comparison with age and sex-matched controls. The
significance of these observations is further supported by a recent
report demonstrating CTGF expression in a screen of human renal
diseases including diabetic nephropathy (Ito, Y., et al. (1998)
Kidney Int. 53, 853-861).
EXAMPLE 4
Induction of Mesangial Cell CTGF Expression by High Glucose
Involves TGF-.beta.1 Dependent and Independent Pathways
[0100] It has been shown that TGF-.beta.1 is a stimulus for
mesangial matrix accumulation in diabetic nephropathy. In our
experimental model as described in Example 1, high glucose
concentrations provoked induction of TGF-.beta.1 mRNA expression in
cultured human mesangial cells over the same temporal framework as
CTGF expression (data not shown).
[0101] To assess the role of TGF-.beta.1 as a stimulus for CTGF
expression in response to high glucose, cells were incubated in
either 5 mM glucose or 30 mM glucose plus 1 .mu.l/ml
anti-TGF-.beta.1 antibody for seven days with three changes of
medium. Cells were serum starved for 24 hr in RPMI 1640 and 0.5%
FBS. 10 ng/ml TGF-.beta.1 (Calbiochem) or 10 ng/ml TGF-.beta.1
preadsorbed with 1 .mu.g/ml neutralising anti-TGF-.beta.1
polyclonal antibody were subsequently added for 24 hr.
[0102] The role of PKC on CTGF expression in response to high
glucose was investigated by culturing the mesangial cells in either
5 mM, 30 mM glucose or 30 mM glucose and the PKC inhibitor GF
102903X.
[0103] FIG. 7 is an autoradiograph of CTGF mRNA levels analysed by
Northern Blot and depicts the results obtained when mesangial cells
were exposed to TGF-.beta.1 (10 ng/ml) for 24 hr in the presence
(lane 3) and absence (lane 2) of anti-TGF-.beta.1 neutralising
antibody (1 .mu.g/ml). Cells cultured in RPMI 1640 and 0.5% FBS for
24 hr served as control (lane 1). A 2.4 kb band was detected
following hybridisation to the CTGF probe. The blot was stripped
and reprobed with GAPDH. The relative amount of CTGF mRNA as
estimated by Phosphor Imager quantification (FIG. 8). Values were
normalised to GAPDH levels and the results are representative of
two independent experiments.
[0104] These results indicate that TGF-.beta.1 is a potent inducer
of increased CTGF mRNA levels under these conditions. This effect
was inhibited by the addition of a neutralising anti-TGF-.beta.1
antibody as depicted in FIG. 7.
[0105] FIG. 9 is an autoradiograph of CTGF mRNA levels analysed by
Northern Blot and depicts the results obtained when mesangial cells
were exposed to 5 mM glucose (lane 1), 30 mM glucose (lane 2) and
30 mM glucose in the presence of anti-TGF-.beta.1 neutralising
antibodies (1 .mu.g/ml) (lane 3) for seven days. A 2.4 kb band was
detected following hybridisation to the CTGF probe. The blot was
stripped and probed with GAPDH. The relative amount of CTGF mRNA as
estimated by Phosphor Imager quantification (FIG. 10). Values were
normalised to GAPDH levels.
[0106] The neutralising anti-TGF-.beta.1 antibody partially
attenuated the glucose-induced increase in CTGF transcript level in
mesangial cells grown in 30 mM glucose for 7 days (FIG. 9),
suggesting that high glucose triggers mesangial cell CTGF
expression through TGF-.beta.1-dependent and independent
pathways.
[0107] FIG. 11 is an autoradiograph of CTGF mRNA levels analysed by
Northern Blot and depicts the results obtained when mesangial cells
were exposed to 5 mM glucose (lane 1), 30 mM glucose (lane 2) and
30 mM glucose in PKC inhibitor GF102903X (10 .mu.M) (lane 3) for
four days. A 2.4 kb band was detected following hybridisation to
the CTGF probe. The blot was stripped and probed with GAPDH. The
relative amount of CTGF mRNA as estimated by Phosphor Imager
quantification (FIG. 12). Values were normalised to GAPDH
levels.
[0108] Whereas the PKC inhibitor GF102903X was without effect on
TGF-.beta.1-induced CTGF expression in our system (data not shown),
this compound afforded partial inhibition of high glucose-induced
CTGF expression (FIG. 11).
[0109] CTGF shares some of the biological actions of TGF-.beta. I
such as stimulation of cell proliferation and extracellular matrix
protein synthesis in fibroblasts. When considered in this context,
our results suggest that TGF-.beta.1 may promote mesangial matrix
production, in part, by inducing CTGF synthesis. TGF-.beta.1 has a
complex profile of biological activities that includes
pro-inflammatory, pro-fibrotic and anti-inflammatory effects. By
targeting CTGF it may be possible to attenuate the
sclerosis-inducing effects of TGF-.beta.1 while preserving its more
desirable anti-inflammatory activities.
EXAMPLE 5
Further Characterisation of IHG-2
[0110] IHG-2 is a mesangial cell gene which we have identified as
being induced in human mesangial cells by high extracellular
glucose as described in Example 1. To further characterise this
gene, IHG-2 was searched against the dbEST using the BLAST
algorithm. This search identified a clone that was 94% identical to
ESTAA071138, clone no: 530117 3'. The sequence for the 5' end of
this clone was also in the database, which again identified
multiple ESTs. These ESTs showed homology with the 3' untranslated
region (UTR) of a rat cDNA clone known as drm/Gremlin. As indicated
above, gremlin/drm, together with DAN and cerberus, are members of
the cysteine knot super-family which includes TGF.beta. and bone
morphogenetic protein (BMP). A second EST W48852, clone no:324951
3', was identified from the IHG-2 BLAST. The 5' end of this clone,
EST W48619, was also searched against the database, from which EST
AA373348 was obtained. This clone showed homology with the drn 3'
UTR, approximately 500 bp from the open reading frame (ORF). Thus,
it was possible to make a direct link from IHG-2 to within 500 bp
of the ORF of drm/gremlin. Therefore, by establishing a link
between EST AA37348 and the ORF of dmm/gremlin, it was confirmed
that IHG-2 is part of the 3' UTR of this gene. Primers were
designed to recognise the ORF, IHG-2, and the EST clone AA373348.
An initial PCR using primers corresponding to the start site of the
gremlin/drm gene together with a primer within the IHG-2 clone
would give a predicted product of approximately 2.5 kb. This
product was nested with primers corresponding to the 3' end of the
ORF of gremlin and the EST clone AA373348, generating a product of
approximately 500 bp, thus verifying that this EST is in the UTR of
the human drm/gremlin gene. Therefore, IHG-2 was found to be part
of the drm/gremlin gene, which was not previously known (FIG.
13).
EXAMPLE 6
Use of Cloning In-Silico Coupled with PCR to Demonstrate that IHG-2
is Part of the 3' Untranslated Region of Gremlin
[0111] In Example 5 we describe the identification of a transcript,
IHG-2, the sequence of which did not show homology against the
cumulative database of characterised sequences using the BLAST
algorithm.
[0112] Bioinformatic analysis was carried out as follows:
[0113] Database searching and alignments were performed at the
National Center for Biotechnology Information (NCBI) Bethesda, Md.,
U.S.A. using the Basic Local Alignment Search Tool Algorithm
(BLAST) (Altschul, S. F., et al. (1997) Nucleic Acids Res. 25,
3389-3402). The Non Redundant (nr) and the Expressed Sequence Tag
(EST) databases were sourced. Contiguous sequences were generated
using Fragment Assembly, a program within the Genetics Computer
Group Inc. package. UniBlast (Guffanti, A., and Simon, G., Trends
in Genetics, 14, 293) was used to identify homologous clusters
within the UniGene database and to verify the consensus sequence
derived from ESTs. Chromosomal localization data were obtained from
the UniGene and Online Mendelian Inheritance in Man (OMIM)
databases.
[0114] To further characterise the sequence, the sequence of IHG
was searched against the EST database where a number of matches
were obtained. Each of these matches was, in turn, searched against
the nr database at NCBI. Four ESTs, namely W52686, N28395, H80042,
and W47324, showed low homology to the 3' UT region of the rat gene
drm referred to in Example 5. The ORF of the human homologue of
this gene, gremlin, was also in the database.
[0115] To generate a link between the ORF of gremlin and IHG-2,
successive BLAST searches were used to identify overlapping
sequences in the EST database.
[0116] However, it was not possible to directly link IHG-2 to the
ORF of gremlin with sequences within the EST database. Therefore,
RNA isolated from human mesangial cells was reverse transcribed
with a primer that recognises IHG-2. PCR analyses were performed
spanning the regions shown in FIG. 13.
[0117] In this graphical representation of a BLAST output from the
EST database, the thick bar of 4 kb represents the final composite
sequence of the gremlin gene. Each of the other bars represents an
individual sequence, in the EST database, that were assembled,
where possible, into continuous sequences, and demonstrates how
cloning in-silico was used to generate the contiguous sequence. The
region of no EST overlap was generated by reverse transcription of
human mesangial mRNA with a complementary primer to IHG-2. PCR was
performed spanning the regions indicated (by arrows), and the
resulting products were sequenced, allowing a contiguous cDNA of
4049 bp to be generated which included the open reading frame of
gremlin and IHG-2.
[0118] Human gremlin and rat drm cDNAs were compared and FIG. 14
shows a graphical representation of an alignment between rat drm
and human gremlin together with the region corresponding to IHG-2
using the BLAST algorithm. Sequence homology was found to be high
in the coding region of the cDNA; however, there are only small
regions of homology within the 3' UT region. This explains why
IHG-2 did not identify drm in a BLAST search, but a match to drm,
and thus gremlin, was obtained by examining EST sequences further.
The lack of homology between rat drm and human gremlin probably
results from decreased selective pressure on the 3' UT region of
gene homologues to remain the same between species.
[0119] FIG. 15 shows the final sequence of human mesangial cell
gremlin, indicating the ORF and the region corresponding to IHG-2
(GenBank accession no: AF 110137). Shown are the 5' and 3' UT
sequence, and the open reading frame (with translation). The boxed
region corresponds to the location of IHG-2. At the time of
submission, this sequence matched 136 separate EST entries in the
EST database. Of these entries, 23.5% were derived from fibroblast
libraries; 30% were from bone tissue libraries; and 34% were
derived from tumor related libraries. The sequence was also
searched against the UniGene database using the UniBlast program.
This identified 4 UniGene clusters, Hs.214148, Hs.40098, Hs.114330,
and Hs.239507. Two of these clusters, Hs.40098, and Hs.239507, have
been mapped to intervals D15S118-144 and D15S144-165 on chromosome
15, respectively. Secretory granule neuroendocrine protein I and
the alpha polypeptide of the nicotinic cholinergic receptor have
been mapped to either side of these clusters and have also been
mapped more specifically to the 15q11-15 interval. Analysis of the
OMIM database reveals that the formin gene, which was recently
shown to induce gremlin expression in the developing limb bud
(Zuniga, A., et al. (1999) supra), is also localised to this
interval. Diabetes mellitus with multiple epiphyseal dysplasia, or
Wolcott-Rallison syndrome, is localised to the 15q11-12 interval
(Stewart, F. J., et al., (1996) Clin. Genet. 49, 152-5). In the
epiphyseal growth plate, immunohistochemical studies have revealed
that BMP-2 and 4 are expressed in proliferating and maturing
chrondocytes, suggesting that BMP and its receptors play roles in
the multi-step cascade of enchondral ossification (Yazaki, Y., et
al., (1998) Anticancer Res. 18, 2339-44). Regulation of gremlin
expression may have implications in both of the disease states
associated with Wolcott-Rallison syndrome.
EXAMPLE 7
Induction of Mesangial Cell Gremlin Expression In Vitro by High
Glucose and Cyclic Mechanical Strain Induce
[0120] A) Primary cultures of human mesangial cells were propagated
as described in Example 1, except that the medium was supplemented
with 5% FBS. Treatment of cultures with glucose was carried out as
described in Example 1. Mesangial cells were exposed to either Sm M
glucose or 30 mM glucose for seven days.
[0121] Northern Blot analysis as described further below was
performed on RNA extracted from mesangial cells grown in either 5
mM (`normal`) or 30 mM (`high`) glucose using the ORF of gremlin
and IHG-2 probes. Both probes detected a 2-fold increase in gene
expression under high glucose conditions as depicted in FIG.
16.
[0122] All subsequent northern analysis was performed using the ORF
of gremlin as a probe.
[0123] Northern Blots were performed using formaldehyde
denaturation according to standard protocols and quantitated using
a phosphorimager (Biorad). PCR products used to generate the probes
for northern analysis were amplified using primers for the open
reading frame (ORF) of gremlin (sense: ATGAGCCGCACAGCCTACAC (SEQ ID
NO: 17); antisense TTAATCCAAATCGATGGATATGC (SEQ ID NO: 18)), and
for IHG-2 (sense: CTCAGCCTCCTAGCCAAGTCC (SEQ ID NO 19); antisense:
GTATTGTCCACATTCTCCAAC (SEQ ID NO: 20)). Fibronectin and GAPDH
probes were generated as described in Example 2
[0124] Specific primers were used to amplify gremlin/IHG-2
(external sense: ATGAGCCGCACAGCCTACAC (SEQ ID NO: 21); external
antisense: GTATTGTCCACATTCTCCAAC (SEQ ID NO: 22); internal sense:
GAGAGTCACACGTGTGAAGC (SEQ ID NO: 23); internal antisense:
AGGAGGATGCAAGCACAGG (SEQ ID NO: 24), BMP-2 (external sense:
CGCGGATCCTGCTTCTTAGACGGACTGCG (SEQ ID NO: 25); external antisense:
TTTGCTGTACTAGCGACACC (SEQ ID NO: 26); internal sense:
CAAGATGAACACAGCTGG (SEQ ID NO 27)), and GCTCAGGATACTCAAGAC (SEQ ID
NO: 28)). RT-PCR was carried out as reported as described in
Example 3.
[0125] The results are shown in FIGS. 16 and 17.
[0126] In FIGS. 16 and 17, lane 1 corresponds to the mesangial
cells exposed to 5 mM glucose and lane 2 corresponds to the
mesangial cells exposed to 30 mM glucose.
[0127] FIG. 16 is an autoradiograph of IHG-2 (1), gremlin (2),
fibronectin (3) and GAPDH (4) mRNA levels analysed by Northern
Blot. Two bands of approximately 4.4 kb and 4.6 kb were detected
following hybridisation to the gremlin and IHG-2 probes.
[0128] FIG. 17 depicts relative mRNA levels as estimated by
Phosphor Imager quantification. Values were normalised to GAPDH
levels. The results are representative of three independent
experiments.
[0129] B) Glomerular hypertension is an independent risk factor for
the development of glomerulosclerosis in diabetes mellitus
(Brenner, B. M., et al. (1982) N. Engl. J. Med. 307, 652-9). To
model the effects of glomerular hypertension on mesangial cell
gremlin expression in the present study, mesangial cells were
propagated under conditions of cyclic mechanical strain for 24 and
48 h in the Flexercell.TM. System
[0130] For the application of mechanical cyclic stretch, primary
human mesangial cells were seeded on either flexible or rigid
based, elastin coated six-well plates (Flex I and Flex II plates,
Flex CelI.TM. Int, Hillsborough, N.H., USA). Cells were grown to
90% confluency, then serum restricted in Clonetics.TM. Mesangial
Basal Medium supplemented with 0.5% fetal calf serum. Cells
cultured on flexible plates were subjected to repeated cycles of
computer-controlled, vacuum-driven mechanical stretch and
relaxation using the Flexercell Strain Unit FX-2000
(Flexercell.TM.). Cells were alternately stretched and relaxed at
0.5 sec intervals (60 cycles/min) for either 24 or 48 h. The
applied vacuum achieved a 17% elongation of the outer annulus of
the culture plates. All experiments were carried out at 37.degree.
C. and 5% CO.sub.2 in a humidified incubator.
[0131] The results were obtained from three diabetic rats, 14 weeks
following onset of diabetes and from three age matched
controls.
[0132] The model used perturbs mesangial cell matrix production and
metabolism in a manner similar to that observed in diabetic
glomerulosclerosis in vivo. Mesangial cell gremlin mRNA levels were
significantly enhanced under conditions of mechanical strain, in
parallel with increased fibronectin mRNA expression as shown in
FIGS. 18 and 19.
[0133] Referring to FIGS. 18 and 19 mesangial cells in culture were
grown under static conditions (lane 1) or during exposure to cyclic
stretch for 24 h (lane 2) or 48 h (lane 3) using the Flexercell.TM.
System.
[0134] FIG. 18 is an autoradiograph of gremlin(1), fibronectin(2)
and GAPDH(3) mRNA analysed by Northern Blot.
[0135] In FIG. 18 the lanes represent the following:
[0136] Lane 1: Mesangial cells grown in culture under static
conditions;
[0137] Lane 2: Mesangial cells grown in culture during exposure to
cyclic stretch for 24 h; and
[0138] Lane 3: Mesangial cells grown in culture during exposure to
cyclic stretch for 48 h.
[0139] FIG. 19 depicts relative mRNA levels as estimated by
Phosphor Imager quantification. Values were normalised to GAPDH
levels.
[0140] To assess gremlin expression in diabetic nephropathy in
vivo, gremlin mRNA levels were measured by Northern Blot analysis
with RNA isolated from the renal cortex of control rats and from
diabetic rats 14 weeks after induction of diabetes mellitus by
streptozotocin (STZ). In keeping with the in vitro experiments
reported above, gremlin mRNA levels were increased in kidneys from
diabetic rats, coincident with proteinuria and histologic evidence
of diabetic nephropathy (data not shown).
[0141] Nine Male Munich-Wistar uninephrectomized rats were rendered
diabetic with streptozotocin, and the RNA extracted as described in
Example 3.
[0142] The results are shown in FIGS. 20 and 21, which show gremlin
mRNA levels in renal cortex of STZ-diabetic rats.
[0143] FIG. 20 is an autoradiograph of gremlin mRNA levels in the
kidney cortex of a STZ-diabetic rat (lane 2) and an age matched
control (lane 1) analysed by Northern Blot. A band of approximately
4.4 kb was detected following hybridisation to the gremlin
probe.
[0144] FIG. 21 depicts relative mRNA levels as estimated by
Phosphor Imager quantification. Values were normalised to GAPDH
levels.
EXAMPLE 8
Regulation of Mesangial Cell Gremlin Expression by High Glucose:
Evidence for Involvement of TGF-.beta.1
[0145] As indicated above, both high ambient glucose concentrations
and cyclic mechanical strain provoke TGF-.beta.1 production by
mesangial cells in vitro and TGF-.beta.1 appears to be a major
stimulus for mesangial matrix accumulation in diabetic glomeruli in
vivo. To probe the mechanism by which high glucose triggers gremlin
expression, primary human mesangial cells were propagated in 5 mM
or 30 mM glucose in the presence and absence of anti-TGF-.beta.1
neutralising antibody (1 .mu.g/ml). Treatnent of cultures with
glucose and anti-TGF-.beta.1 were as described in Example 1 and
Example 4, respectively. To assess the role of TGF-.beta.1 as a
stimulus for gremlin expression, cells were serum restricted for 24
h in MCDB 131 and 0.5% FBS and subsequently treated with 10 ng/ml
TGF-.beta.1. MCDB131 is a specialised medium for the growth of
mesangial cells and is obtained from Clonetics.
[0146] Initial studies had indicated that TGF-.beta.1 neuturalizing
antibody (data not shown) blunted glucose-triggered gremlin
expression and therefore the ability of TGF-.beta.1 to alter
gremlin expression was investigated. The results are shown in FIGS.
22 and 23.
[0147] The addition of exogenous human recombinant TGF-.beta.1 (10
ng/ml, 24 h) to serum restricted (24 h) mesangial cells also
augmented gremlin mRNA levels, suggesting that high glucose
enhances gremlin mRNA expression, at least in part, through its
ability to stimulate TGF-.beta.1 expression. In aggregate, these
observations suggest the presence of a novel autocrine loop through
which TGF-.beta.1 induces gremlin gene expression and may thereby
regulate the activity of mesangial-derived BMPs as hereinafter
described.
[0148] It was found that gremlin expression in response to high
glucose (30 mM, 7 days) was reduced in the presence of
anti-TGF-.beta.1 antibody (data not shown). To further probe the
role of TGF-.beta.1 as a modulator of gremlin expression, mesangial
cells were exposed to TGF-.beta.1 (10 ng/ml) for 24 h (lane 2).
Cells cultured in MCDB131 and 0.5% FBS for 24 h served as a control
(lane 1).
[0149] FIG. 22 is an autoradiograph of gremlin (1), fibronectin (2)
and GAPDH (3) mRNA levels analysed by Northern Blot.
[0150] FIG. 23 shows relative mRNA levels as estimated by Phosphor
Imager quantification. Values were normalised to GAPDH levels.
EXAMPLE 9
High Glucose Stress Induces BMP-2, but not BMP-4 Expression in
Mesangial Cells
[0151] As indicated above gremlin is a putative antagonist of BMP-2
and BMP-4. Specifically, gremlin has been recently reported to form
heterodimers with BMPs and thereby antagonise BMP signalling (Hsu,
D. R., et al. (1998) supra). In the present study, RT-PCR was
employed as an initial assessment of mesangial cell BMP
expression.
[0152] As an initial assessment of the relationship between gremlin
expression and BMP expression, RNA was isolated from mesangial
cells grown for 7 days in either 5 mM or 30 mM glucose. Following
reverse transcription with random primers, a primary PCR of the ORF
of BMP-2 was performed. This product, which was undetectable on an
ethidium stained agarose gel after 30 cycles, was nested to give a
predicted product of 446 bp. PCR analysis with BMP-4 and GAPDH
specific primers gave predicted products of 378 bp and 452 bp
respectively.
[0153] FIG. 24 depicts representative reactions of 4 independent
experiments. 10 .mu.l of each PCR reaction was run on 1% ethidium
bromide stained agarose gels.
[0154] Whereas little or no BMP-2 mRNA was detected in mesangial
cells propagated in 5 mM glucose, a marked induction of BMP-2
expression was observed in cells cultured in 30 mM glucose as shown
in FIG. 24.
[0155] In contrast, BMP-4 expression levels were relatively
unchanged. Interestingly, whereas BMP-2 does not stimulate
fibronectin expression in mesangial cells in vitro, BMP-2 has been
recently shown to block mesangial cell proliferation triggered by
epidermal growth factor and platelet-derived growth factor (Ghosh
Choundhury, G., et al., (1999) J. Biol. Chem. 274, 10897-902; Ghosh
Choudhury, G., et al., (1999) Biochem. Biophys. Res. Commun. 258,
490-6). Our results raise the possibility that TGF-.beta.1
stimulated expression of gremlin may contribute to mesangial cell
proliferative responses in this setting. The influence of gremlin
on cell proliferation appears complex, however, and may vary
markedly depending on the cell-type and proliferative stimulus. In
contrast to the aforementioned potentially pro-proliferative
actions, over-expression of the gremlin homologue, drm, causes
apoptosis in fibroblasts by an ERK mediated pathway, while cells
transformed with oncogenes such as v-mos show suppressed drn
expression (Topol, L. Z., et al., (1997) Mol. Cell. Biol. 17,
4801-10). Similarly, over-expression of DAN, another cysteine knot
super-family member with homology to drm/gremlin, retards
fibroblast entry into S phase (Ozaki, T., et al., (1995) Cancer
Res. 55, 895-900).
[0156] In summary, our results demonstrate that the DAN family
member gremlin is induced in diabetic nephropathy in vivo and
implicate both metabolic and hemodynamic stress as stimuli for
gremlin expression.
[0157] The findings that high glucose-triggered gremlin expression
is mimicked by addition of exogenous TGF-.beta.1, blunted by
anti-TGF-.beta.1 neutralising antibody, and occurs in association
with induction of mesangial cell BMP-2 suggests the presence of a
novel autocrine loop which may limit the bioactivity of TGF-.beta.1
superfamily members and modulate mesangial cell proliferation
within the diabetic mesangium. The further elucidation of the
functional interactions of the DAN family of secreted proteins,
such as gremlin, with TGF-.beta.1 superfamily members may shed
light on the complex multi-pronged molecular events that perturb
cell proliferation and matrix production in diabetic
glomerulosclerosis.
Sequence CWU 1
1
33 1 598 DNA Homo sapiens Unsure (1)..(598) any n =a,c,g,t any
unknown or other 1 ttggaatagt tcttgcttta taaaaatagt actgcgatta
aaaaaaaagc acttctgcca 60 aaggaaccat gttccaacac cgcaaacaag
gtgttctgct taaacagagt aagatacacc 120 acccccatcc atcccttcct
tccctgttcc cctcccaact tgagttgtgt cattcgcacc 180 agtgtcctgg
gtggtaggga tgctacagcc acctaaggca aggagccctg ggaggtggga 240
gggcttgcat ggttaagcac accagaactg aagcgcaaaa gggtcagctg tcttcatcta
300 gaatctctgg atgttccttc cagaaagcat ccccgatgat atcgcagtgc
aagggcactg 360 gctttgtcct ggtccgggtc actgccatct tttttccttc
catttctgtt ggcagcttaa 420 tttcttttgt catcacttca tccaccttct
gccatatcaa cacagtccct ttcctataca 480 tcggcagctc attattatag
ttgatgttga attcagaaaa caaaatctca ttcttgtctg 540 ctgnaagagt
tccctgtaat ctcccttggg cttgtactgg tgttagtcca gattgttg 598 2 761 DNA
Homo sapiens Unsure (1)..(761) any n =a,c,g,t any unknown or other
2 ggtcctttaa agtctggttg ctgggataca ccacgactct tccggtcaaa gcctggggga
60 tacagaaggg gctrgtcctc aaagtaatcc cgccaataaa acayatagct
ggaggcaaac 120 tgggaggyca cgtgagtcat gaactttact ggctcttctt
ttaaaccaat tggttttccg 180 cttgwacaca aagctgtact catcactctg
tccataacgc gatcacaata tcctctagtt 240 cttccatcac agtctgcgca
catttggtca tcagctggag agcacggctg tcattgggtt 300 ttgcaaagtt
gtgcttctca gcaaaccgat ggaaattccg gccgtccagc cgnactacca 360
cccagcagtg tgccaggcag gtgtcgtcag cctcgaagtc cctcacgtac tcgaacttgc
420 tttttgccat ggtcgccccc aatctcaggt accgtctcag agtgatggaa
atggtggcca 480 aggaatcgtg aaccttaact ttacaggcgc cccacattct
acacgcggaa aggaaagggc 540 cagatagccc cgccccggaa gtgttctctt
cgtggctact ctagccgtag ggcggtcata 600 gtctctctcg sctctccctg
kagttcttaa mcyyccaggg aaaraggatg gaggtttagg 660 ttcctccgtt
agcaccttcc acgcttgctt cttcctcctc ccggtctgcg gcaaatcagt 720
ctcacgaggt ttttaaaaat tattttttat ctgctggcct t 761 3 393 DNA Homo
sapiens Unsure (1)..(393) any n =a,c,g,t any unknown or other 3
atgacacaaa tattaggatt ttatttttac tattatccac cagcaacaag atatcaaaca
60 ctggttctgt gattatttaa tggtgaaaaa gttgaataaa tcaatttagt
atacccatat 120 gttggaatat tgagtccatt tttcttttaa aaatcacact
ttggaataat tgatgatact 180 ggcaaatgct caagctgagt ggaaaaatat
ataaacattg tataggcgaa taattccaat 240 cttgtgcatt ccctgtgtaa
acctacatac acaaaaagaa aaaagactga aaggaaccat 300 ccacaatgct
ttgatcggga aagacggaga aacaaagtgt taattttctt aactatagtt 360
ttnggtgtat tccagatttt ctacaagtta ata 393 4 435 DNA Homo sapiens 4
gtactttgga tttggttaac ctgttttctt caagcctgag gttttatata caaactccct
60 gaatactctt tttgccttgt atcttctcag cctcctagcc aagtcctatg
taatatggaa 120 aacaaacact gcagacttga gattcagttg ccgatcaagg
ctctggcatt cagagaaccc 180 ttgcaactcg agaagctgtt tttatttcgt
ttttgttttg atccagtgct ctcccatcta 240 acaactaaac aggagccatt
tcaaggcggg agatatttta aacacccaaa atggttgggt 300 ctgattttca
aacttttaaa attcactact gatgattctg cacgctaagg cgaatttggt 360
ccaaacacat aagtgtgtgt gttttgtata cactgtatga ccccacccca aatctttgta
420 ttgtccacat tctcc 435 5 273 DNA Homo sapiens Unsure (1)..(273)
any n =a,c,g,t any unknown or other 5 agaagcaatt taggaanccn
acagnaaana aatgctgttt tataggagag aaaacacggc 60 acaccaaggt
taagtagttt gtagacgatg ttgaataggt tcaggtacag gtcaatgcag 120
tgatgaggaa agcacctang tatacttgac agatagtccc ctttgcttaa cacccaactc
180 ctccaccctg tgcagtttnn cttgtgccag tgatcacagg attcgctgag
tgaattacca 240 taattggatt taattcacga aggggatgtt ttc 273 6 309 DNA
Homo sapiens 6 attgatagag gccctgtttc atgacatttc atgagtttca
atatgttgtt cagcatgttg 60 tgaggtgact ctcagcccct ttcccactga
gatggactgt ggtgatgctg tgagggtgtg 120 actgacacac cttcatgtgc
ccaagcatgg gtttgatcac aggtcacatg cagtttttgg 180 catagtaaat
gtatcattgt tcttttcctc cctcctaaag gaaacagagg aatccacctg 240
tatgagagtg ccatgtaggg ataaacttaa aggacagatg acacattggt catgttcgtg
300 ataaggaaa 309 7 20 DNA Homo sapiens 7 accacagtcc atgccatcac 20
8 20 DNA Homo sapiens 8 tccaccaccc tgttgctgta 20 9 20 DNA Homo
sapiens 9 ggtcttcctg gcttaaaggg 20 10 20 DNA Homo sapiens 10
gctggtcagc cctgtagaag 20 11 20 DNA Homo sapiens 11 ccaggagttc
caggatttca 20 12 20 DNA Homo sapiens 12 ttttggtccc agaaggacac 20 13
18 DNA Homo sapiens 13 cgaaatcaca gccagtag 18 14 18 DNA Homo
sapiens 14 atcacatcca cacggtag 18 15 20 DNA Homo sapiens 15
ctaagacctg tggaatgggc 20 16 21 DNA Homo sapiens 16 ctcaaagatg
tcattgtccc c 21 17 20 DNA Homo sapiens 17 atgagccgca cagcctacac 20
18 23 DNA Homo sapiens 18 ttaatccaaa tcgatggata tgc 23 19 21 DNA
Homo sapiens 19 ctcagcctcc tagccaagtc c 21 20 21 DNA Homo sapiens
20 gtattgtcca cattctccaa c 21 21 20 DNA Homo sapiens 21 atgagccgca
cagcctacac 20 22 21 DNA Homo sapiens 22 gtattgtcca cattctccaa c 21
23 20 DNA Homo sapiens 23 gagagtcaca cgtgtgaagc 20 24 19 DNA Homo
sapiens 24 aggaggatgc aagcacagg 19 25 29 DNA Homo sapiens 25
cgcggatcct gcttcttaga cggactgcg 29 26 20 DNA Homo sapiens 26
tttgctgtac tagcgacacc 20 27 18 DNA Homo sapiens 27 caagatgaac
acagctgg 18 28 18 DNA Homo sapiens 28 gctcaggata ctcaagac 18 29 341
DNA Rattus sp. 29 atctccaccc gggttaccaa tgacaatact ttctgcaggc
tggagaagca gagtcgtctc 60 tgcatggtca ggccctgtga agctgaccta
gaggaaaaca ttaagaaggg caaaaagtgc 120 atccggacgc ctaaaattgc
caagcctgtc aagtttgagc tttctggctg caccagtgtg 180 aagacctacc
gggctaagtt ctgtggggtg tgcacggacg gccgctgctg cacaccgcac 240
agaaccacca cactgccggt ggagttcaag tgcccccatg gcgaaatcat gaaaaagaac
300 atgatgttca tcaagacctg tgcctgccat tacaactgtc c 341 30 341 DNA
Mus sp. 30 atctccaccc gagttaccaa tgacaatacc ttctgcagac tggagaagca
gagccgcctc 60 tgcatggtca ggccctgcga agctgacctg gaggaaaaca
ttaagaaggg caaaaagtgc 120 atccggacac ctaaaatcgc caagcctgtc
aagtttgagc tttctggctg caccagtgtg 180 aagacataca gggctaagtt
ctgcggggtg tgcacagacg gccgctgctg cacaccgcac 240 agaaccacca
ctctgccagt ggagttcaaa tgccccgatg gcgagatcat gaaaaagaat 300
atgatgttca tcaagacctg tgcctgccat tacaactgtc c 341 31 113 PRT Rattus
sp. 31 Ile Ser Thr Arg Val Thr Asn Asp Asn Thr Phe Cys Arg Leu Glu
Lys 1 5 10 15 Gln Ser Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp
Leu Glu Glu 20 25 30 Asn Ile Lys Lys Gly Lys Lys Cys Ile Arg Thr
Pro Lys Ile Ala Lys 35 40 45 Pro Val Lys Phe Glu Leu Ser Gly Cys
Thr Ser Val Lys Thr Tyr Arg 50 55 60 Ala Lys Phe Cys Gly Val Cys
Thr Asp Gly Arg Cys Cys Thr Pro His 65 70 75 80 Arg Thr Thr Thr Leu
Pro Val Glu Phe Lys Cys Pro His Gly Glu Ile 85 90 95 Met Lys Lys
Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn 100 105 110 Cys
32 113 PRT Mus sp. 32 Ile Ser Thr Arg Val Thr Asn Asp Asn Thr Phe
Cys Arg Leu Glu Lys 1 5 10 15 Gln Ser Arg Leu Cys Met Val Arg Pro
Cys Glu Ala Asp Leu Glu Glu 20 25 30 Asn Ile Lys Lys Gly Lys Lys
Cys Ile Arg Thr Pro Lys Ile Ala Lys 35 40 45 Pro Val Lys Phe Glu
Leu Ser Gly Cys Thr Ser Val Lys Thr Tyr Arg 50 55 60 Ala Lys Phe
Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro His 65 70 75 80 Arg
Thr Thr Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Ile 85 90
95 Met Lys Lys Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn
100 105 110 Cys 33 4049 DNA Homo sapiens 33 gcggccgcac tcagcgccac
gcgtcgaaag cgcaggcccc gaggacccgc cgcactgaca 60 gtatgagccg
cacagcctac acggtgggag ccctgcttct cctcttgggg accctgctgc 120
cggctgctga agggaaaaag aaagggtccc aaggtgccat ccccccgcca gacaaggccc
180 agcacaatga ctcagagcag actcagtcgc cccagcagcc tggctccagg
aaccgggggc 240 ggggccaagg gcggggcact gccatgcccg gggaggaggt
gctggagtcc agccaagagg 300 ccctgcatgt gacggagcgc aaatacctga
agcgagactg gtgcaaaacc cagccgctta 360 agcagaccat ccacgaggaa
ggctgcaaca gtcgcaccat catcaaccgc ttctgttacg 420 gccagtgcaa
ctctttctac atccccaggc acatccggaa ggaggaaggt tcctttcagt 480
cctgctcctt ctgcaagccc aagaaattca ctaccatgat ggtcacactc aactgccctg
540 aactacagcc acctaccaag aagaagagag tcacacgtgt gaagcagtgt
cgttgcatat 600 ccatcgattt ggattaagcc aaatccaggt gcacccagca
tgtcctagga atgcagcccc 660 aggaagtccc agacctaaaa caaccagatt
cttacttggc ttaaacctag aggccagaag 720 aacccccagc tgcctcctgg
caggagcctg cttgtgcgta gttcgtgtgc atgagtgtgg 780 atgggtgcct
gtgggtgttt ttagacacca gagaaaacac agtctctgct agagagcact 840
ccctattttg taaacatatc tgctttaatg gggatgtacc agaaacccac ctcaccccgg
900 ctcacatcta aaggggcggg gccgtggtct ggttctgact ttgtgttttt
gtgccctcct 960 ggggaccaga atctcctttc ggaatgaatg ttcatggaag
aggctcctct gagggcaaga 1020 gacctgtttt agtgctgcat tcgacatgga
aaagtccttt taacctgtgc ttgcatcctc 1080 ctttcctcct cctcctcaca
atccatctct tcttaagttg atagtgacta tgtcagtcta 1140 atctcttgtt
tgccaaggtt cctaaattaa ttcacttaac catgatgcaa atgtttttca 1200
ttttgtgaag accctccaga ctctgggaga ggctggtgtg ggcaaggaca agcaggatag
1260 tggagtgaga aagggagggt ggagggtgag gccaaatcag gtccagcaaa
agtcagtagg 1320 gacattgcag aagcttgaaa ggccaatacc agaacacagg
ctgatgcttc tgagaaagtc 1380 ttttcctagt atttaacaga acccaagtga
acagaggaga aatgagattg ccagaaagtg 1440 attaactttg gccgttgcaa
tctgctcaaa cctaacacca aactgaaaac ataaatactg 1500 accactccta
tgttcggacc caagcaagtt agctaaacca aaccaactcc tctgctttgt 1560
ccctcaggtg gaaaagagag gtagtttaga actctctgca taggggtggg aattaatcaa
1620 aaacckcaga ggctgaaatt cctaatacct ttcctttatc gtggttatag
tcagctcatt 1680 tccattccac tatttcccat aatgcttctg agagccacta
acttgattga taaagatcct 1740 gcctctgctg agtgtacctg acagtaagtc
taaagatgar agagtttagg gactactctg 1800 ttttagcaag aratattktg
ggggtctttt tgttttaact attgtcagga gattgggcta 1860 ragagaagac
gacgagagta aggaaataaa gggrattgcc tctggctaga gagtaagtta 1920
ggtgttaata cctggtagaa atgtaaggga tatgacctcc ctttctttat gtgctcactg
1980 aggatctgag gggaccctgt taggagagca tagcatcatg atgtattagc
tgttcatctg 2040 ctactggttg gatggacata actattgtaa ctattcagta
tttactggta ggcactgtcc 2100 tctgattaaa cttggcctac tggcaatggc
tacttaggat tgatctaagg gccaaagtgc 2160 agggtgggtg aactttattg
tactttggat ttggttaacc tgttttcttc aagcctgagg 2220 ttttatatac
aaactccctg aatactcttt ttgccttgta tcttctcagc ctcctagcca 2280
agtcctatgt aatatggaaa acaaacactg cagacttgag attcagttgc cgatcaaggc
2340 tctggcattc agagaaccct tgcaactcga gaagctgttt ttatttcgtt
tttgttttga 2400 tccagtgctc tcccatctaa caactaaaca ggagccattt
caaggcggga gatattttaa 2460 acacccaaaa tgttgggtct gattttcaaa
cttttaaact cactactgat gattctcacg 2520 ctaggcgaat ttgtccaaac
acatagtgtg tgtgttttgt atacactgta tgaccccacc 2580 ccaaatcttt
gtattgtcca cattctccaa caataaagca cagagtggat ttaattaagc 2640
acacaaatgc taaggcagaa ttttgagggt gggagagaag aaaagggaaa gaagctgaaa
2700 atgtaaaacc acaccaggga ggaaaaatga cattcagaac cagcaaacac
tgaatttctc 2760 ttgttgtttt aactctgcca caagaatgca atttcgttaa
tggagatgac ttaagttggc 2820 agcagtaatc ttcttttagg agcttgtacc
acagtcttgc acataagtgc agatttggct 2880 caagtaaaga gaatttcctc
aacactaact tcactgggat aatcagcagc gtaactaccc 2940 taaaagcata
tcactagcca aagagggaaa tatctgttct tcttactgtg cctatattaa 3000
gactagtaca aatgtggtgt gtcttccaac tttcattgaa aatgccatat ctataccata
3060 ttttattcga gtcactgatg atgtaatgat atattttttc attattatag
tagaatattt 3120 ttatggcaag atatttgtgg tcttgatcat acctattaaa
ataatgccaa acaccaaata 3180 tgaattttat gatgtacact ttgtgcttgg
cattaaaaga aaaaaacaca catcctggaa 3240 gtctgtaagt tgttttttgt
tactgtaggt cttcaaagtt aagagtgtaa gtgaaaaatc 3300 tggaggagag
gataatttcc actgtgtgga atgtgaatag ttaaatgaaa agttatggtt 3360
atttaatgta attattactt caaatccttt ggtcactgtg atttcaagca tgttttcttt
3420 ttctccttta tatgactttc tctgagttgg gcaaagaaga agctgacaca
ccgtatgttg 3480 ttagagtctt ttatctggtc aggggaaaca aaatcttgac
ccagctgaac atgtcttcct 3540 gagtcagtgc ctgaatcttt attttttaaa
ttgaatgttc cttaaaggtt aacatttcta 3600 aagcaatatt aagaaagact
ttaaatgtta ttttggaaga cttacgatgc atgtatacaa 3660 acgaatagca
gataatgatg actagttcac acataaagtc cttttaagga gaaaatctaa 3720
aatgaaaagt ggataaacag aacatttata agtgatcagt taatgcctaa gagtgaaagt
3780 agttctattg acattcctca agatatttaa tatcaactgc attatgtatt
atgtctgctt 3840 aaatcattta aaaacggcaa agaattatat agactatgag
gtaccttgct gtgtaggagg 3900 atgaaagggg agttgatagt ctcataaaac
taatttggct tcaagtttca tgaatctgta 3960 actagaattt aattttcacc
ccaataatgt tctatatagc ctttgctaaa gagcaactaa 4020 taaattaaac
ctattctttc aaaaaaaaa 4049
* * * * *