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Title:
SOX9 AS A TARGET FOR FIBROSIS THERAPY
Document Type and Number:
WIPO Patent Application WO/2008/142442
Kind Code:
A1
Abstract:
Inappropriate expression of the transcription factor SOX9 has been linked to liver fibrosis providing an attractive new target for halting progress of liver fibrosis to cirrhosis. It is extrapolated that abrogation of SOX9 expression may also be used to treat other fibrotic conditions, notably, for example, skin keloid and other skin disorders associated with scarring.

Inventors:
HANLEY NEIL A (GB)
PIPER HANLEY KAREN (GB)
HEALY EUGENE PUIS JOSEPH (GB)
Application Number:
PCT/GB2008/050354
Publication Date:
November 27, 2008
Filing Date:
May 16, 2008
Export Citation:
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Assignee:
UNIV SOUTHAMPTON (GB)
HANLEY NEIL A (GB)
PIPER HANLEY KAREN (GB)
HEALY EUGENE PUIS JOSEPH (GB)
International Classes:
C07K14/47
Other References:
WANG HONGYUN ET AL: "SOX9 is expressed in normal prostate basal cells and regulates androgen receptor expression in prostate cancer cells", CANCER RESEARCH, vol. 67, no. 2, January 2007 (2007-01-01), pages 528 - 536, XP002492407, ISSN: 0008-5472
SONG JASON J ET AL: "Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta 1 to induce mesenchymal cell condensation", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 210, no. 2, February 2007 (2007-02-01), pages 398 - 410, XP002492408, ISSN: 0021-9541
HANLEY KAREN PIPER ET AL: "Ectopic SOX9 mediates extracellular matrix deposition characteristic of organ fibrosis.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 16 MAY 2008, vol. 283, no. 20, 16 May 2008 (2008-05-16), pages 14063 - 14071, XP002492406, ISSN: 0021-9258
Attorney, Agent or Firm:
IRVINE, Jonquil, Claire (4220 Nash CourtOxford, Oxfordshire OX4 2RU, GB)
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Claims:

Claims:

1. An agent capable of specifically reducing upregulated expression of the transcription factor SOX9 for use in treating a condition arising from aberrant extracellular matrix (ECM) component production resulting from such expression, wherein said aberrant ECM component production includes at least typei and / or type 2 collagen.

2. An agent as claimed in claim 1 for use in treating liver fibrosis.

3. An agent as claimed in claim 1 for use in treating a dermal fibrotic disease.

4. An agent as claimed in claim 4 for treating skin keloid.

5. An agent as claimed in claim 1 for treating heart valve disease.

7. An agent as claimed in claim 1 which comprises an entity selected from (i) an siRNA, antisense olignucleotide or antisense PNA which targets SOX9 expression (ii) a vector encoding an oligonucleotide as specified in (i) or a conjugate comprising an entity as specified in (i) conjugated to a second entity for targeting the tissue of interest.

8. An agent as claimed in claim 7 which is in the form of liver-targeting liposomes.

9. An agent as claimed in claim 7 for treatment of a dermal fibrotic condition by topical delivery.

10. A pharmaceutical composition, which comprises an agent as claimed in any one of claims 1 to 9 together with a pharmaceutically acceptable carrier or diluent.

1 1. Use of an agent as claimed in any one of claims 1 to 9 in the manufacture of a medicament for use in treating a condition arising from aberrant ECM component production resulting from SOX9 expression, wherein said aberrant ECM component production includes at least typei and / or type 2 collagen.

12. A use as claimed in claim 11 wherein said condition is selected from liver fibrosis, dermal fibrotic disease and heart valve disease.

13. A method of treating a condition arising from aberrant ECM component production wherein said aberrant ECM component production includes at least typei and / or type 2 collagen, said method comprising administering an agent capable of specifically reducing upregulated expression of the transcription factor SOX9.

14. A method as claimed in claim 14 wherein said condition is selected from liver fibrosis, dermal fibrotic disease and heart valve disease.

15. A method as claimed in claim 13 or claim 14 wherein said agent is an agent as defined in any one of claims 7 to 9.

16. A method of screening for an agent according to claim 1 which comprises contacting cultured cells exhibiting SOX9 expression or reporter gene activation from a SOX9 gene regulatory element- reporter gene construct with an agent and determining whether said reporter gene activation, SOX9 expression, or type 1 and/or type 2 collagen production resulting from such expression, is reduced in said cells by said agent.

17. A method as claimed in claim 16 wherein said cells exhibit SOX9 expression and produce type I and /or type collagen.

18. A method as claimed in claimed in claim 16 wherein said cells are transfected with a S0X9 gene regulatory element - reporter gene construct and treated to activate reporter gene expression.

19. A method as claimed in claim 16 wherein said cells are hepatic stellate cells plated on to a plastic tissue culture plate and exhibit SOX9 expression and collagen production.

Description:

SOX9 As A Target For Fibrosis Therapy

Field of the invention The present invention relates to targeting of expression of the transcription factor SOX9 for treatment of conditions associated with aberrant expression of extracellular matrix (ECM) components resulting from up-regulation of SOX9 such as, for example, expression of collagens and other structural proteins resulting in liver fibrosis.

Background to the invention

The transcription factor SOX9 (sex-determining region Y-box 9) was first identified as having a critical role in normal development of cartilage (chrondrogenesis). It is responsible in chondrocytes for the desposition of ECM predominantly composed of type 2 collagen (COL2) (Bi et al. (1999) Nat. Genet. 22, 85-89). SOX9 directly activates transcription of the COL2A1 gene via a binding site in the first intron (Bell et al. (1997) Nat. Genet. Jj3, 174-178). It is also responsible in chondrocytes for expression of Collagen Oligomeric Protein-1 (COMP1 ) (Liu et al. (2007) Front. Biosci. 12, 3899- 3910). The SOX9 gene has been mapped to chromosome 17 in the same region as the locus for the inherited skeletal disease Campomelic Dysplasia (see Published international Application WO 96/17057). In humans, inactivating mutations of the SOX9 gene cause campomelic dysplasia, a condition characterised by failure of chondrogenesis. SOX9 has also been shown to be involved in the development of the pancreas and neural cell lineages but is inactive during normal liver development.

The mammalian liver and pancreas both develop by budding from the same region of the mammalian foregut according to the balance of fibroblast growth factor signalling. Thereafter, expression of the SOX9 gene becomes restricted to the developing pancreatic progenitors (the inventors' previous work published in Piper et al. Mech. Dev. (2002) 116, 223-6), which give rise to all future pancreatic lineages, and the fetal intestine, eventually localising to the basal stem cell population. In contrast, SOX9 is extinguished in epithelial hepatocytes of the normal developing liver. The inventors have now shown, however, that SOX9 can become ectopically expressed in human fetal hepatocytes via the inhibition of histone deacetylase. This increases the transcription factor Nuclear factor-Y (NF-Y) activation of SOX9 expression with consequent expression of extracellular matrix genes. By means of RNA interference (RNAi), direct association was demonstrated in the same fetal hepatocytes between

SOX9 expression and expression of the type 2 collagen gene COL2A1; type 2 collagen expression was decreased by abrogating SOX9 levels.

In adult liver disease, activated hepatic stellate cells (HSCs) secrete ECM components that cause fibrosis and its end stage, cirrhosis (Iredale J. P. (2007) J. Clin. Invest. 1 17, 539). Normally quiescent in the hepatic sinusoids, HSCs are activated by fibrogenic stimuli, such as chronic Hepatitis B infection in humans or repeated carbon tetrachloride (CCI 4 ) injection in rat models. This is accompanied by development of the spindle-shaped phenotype of a myofibroblast and secretion of copious ECM. In the activated state, HSCs are demarcated by expression of α-smooth muscle actin (αSMA) and type 1 collagen (COL1 ), the latter encoded by the COL1A2 gene. The inventors have now additionally found that SOX9 becomes expressed in HSCs upon activation to produce ECM components. Thus by immunochemistry, nuclear SOX9 was detected within fibrotic regions of rat livers from rats injected with CCI 4 , the liver fibrosis being demonstrated by the deposition of types 1 and 2 collagen. An established model of liver fibrosis uses quiescent HSCs isolated from normal rat livers and plated on to plastic, following which the cells become activated, assuming the myofibroblast appearance and secreting type 1 collagen (Smart et al. (2006) Hepatology 44, 1432- 1440; Iredale J. P. (2007) ibid). The inventors have also now shown that, during this activation process, SOX9 becomes expressed and localises to the nucleus. The activated HSCs were shown to express COL1 and also some COL2 Moreover, abrogation of SOX9 in such activated cells by RNAi was found by the inventors to diminish COL1 expression; reduction of SOX9 level was accompanied by reduction of COL1 by a similar magnitude. The presence of COL2 in activated HSCs was also diminished by RNAi targeting of SOX9 expression.

In vivo, transforming growth factor-β (TGF-β) signalling is a major pro-fibrotic stimulus leading to HSC activation (Iredale J. P. (2007) ibid). The inventors have also more recently studied the effect of TGF-β on SOX9 expression in activated rat primary HSCs in low serum conditions. Interestingly, it was additionally found that TGF-β increased SOX9 expression in such HSCs. Moreover, under similar conditions, SOX9 was also induced approximately 2.5-fold in cells of the human LX2 line, a model for human HSCs

Hence, there is now proposed a new manner of halting progression of liver fibrosis towards cirrhosis reliant upon specifically diminishing upregulated SOX9 expression.

This might be, for example, by directly targeting S0X9 mRNA or transcription of the

S0X9 gene. Furthermore, it is also extrapolated that such targeting of SOX9 expression will have utility in treating other fibrotic conditions in other organs associated with upregulation of SOX9, especially for example skin keloid. Keloid formation represents excessive scarring following trauma to the skin. cDNA microarray analysis and Northern blotting have previously shown that the SOX9 gene is one of many genes upregulated in keloid lesions (Naitoh et al. Genes Cells (2005) W, 1081- 1091 ).

It is also proposed that diminishing SOX9 expression may have utilty in treating other skin diseases associated with scarring, for example in localised scleroderma/morphoea, generalised scleroderma/ systemic sclerosis, acne vulgaris and discoid lupus erythematosus. It may be of interest for preventing hair loss due to scarring alopecias, for example, due to lichen planopilaris, discoid lupus erythematosus and pseudopelade. In addition, the same therapeutic approach is of interest in preventing or reducing scarring of mucosal surfaces (eye, oral cavity/pharynx and oesophagus) in conditions such as cicatricial pemphigoid and systemic sclerosis. Furthermore, it is considered that targeting of SOX9 expression could limit the scarring in inherited skin disorders such as dystrophic epidermolysis bullosa and prevent sequelae such as fusion of the fingers and scarring of the oesophagus. Targeting of SOX9 expression could also reduce scarring of skin which follows third degree burns, scalds and chemical burns.

Dysregulation of ECM organization is also a common feature of heart valve disease and SOX9 has been suggested to have a role in normal remodelling of valve leaflets

(Lincoln et al. Developmental Biol. (2006) 294, 292-302). The observations of the inventors on association between SOX9 expression and liver cell fibrosis therefore also lead to the proposition of targeting of such expression to prevent or treat fibrotic heart valve disease. SOX9 has also been described within the media of calcified vasculature (Neven et al. (2007) Kidney Int. 72, 574-581 ).

More recently, SOX9 was found to activate the enhancer-promoter of the COL4A2 gene and to be increased along with the expression of TGF-β and alpha2 (IV) collagen in mesangial cells in experimental mouse nephrotoxic nephrititis (Sumi et al. Am. J. Pathol. (Epub:2007 April 19);17O, 1854-1864). With the results presented herein, this leads to interest in targeting SOX9 in relation to Type IV collagen deposition associated

with kidney glomerulosclerosis but does not enable extrapolation of utility of SOX9 expression targeting to other fibrotic diseases with a very different aetiology associated with other collagen types, e.g. type I collagen and /or type Il collagen.

Summary of the invention

In one aspect, the present invention therefore provides an agent capable of specifically reducing upregulated expression of the transcription factor SOX9 for use in treating a condition arising from aberrant ECM component production resulting from such expression, wherein said aberrant ECM component production includes at least typei and / or type 2 collagen, especially, for example, liver fibrosis, dermal fibrotic disease, e.g. skin keloid, or cardiac fibrosis such as heart valve disease. In the case of liver fibrosis as noted above both type 1 and type 2 collagen accumulate. Type I collagen is also recognised as a principal component of excessive ECM in skin keloid lesions (Peltonen et al. (1991 ) J. Invest. Dermatol. 97, 240-248; Naitoh et al (2001 ) Biochem. Biophys. Res. Commun.280, 1316-1322; Naitoh et al. ibid). As noted above, transcriptional regulation of the COL2A1 gene by SOX9 is well recognised (Bell et al. (1997) ibid). The type 1 collagen gene COL1A2 has also recently been described as a potential SOX9 target (Ylostalo et al. (2006) Stem Cells 24, 642-652).

Also provided are pharmaceutical compositions comprising such an agent and use of such an agent in the manufacture of a medicament for use in treating a condition arising from aberrant ECM component production as noted above.

In a further aspect, there is provided a method of treating a condition arising from aberrant ECM component production wherein said aberrant ECM component production includes at least typei and / or type 2 collagen, said method comprising administering an agent capable of specifically reducing upregulated expression of SOX9

In a still further aspect, the invention provides a method of screening for an agent for use in treating a condition arising from aberrant ECM component production including at least type 1 and/ or type 2 collagen, e.g. liver fibrosis, which comprises contacting cultured cells exhibiting detectable SOX9 expression or reporter gene activation from a SOX9 gene regulatory element-reporter gene construct with an agent and determining whether said reporter gene activation, SOX9 expression or type 1 and/or type 2 collagen production resulting from such expression, is reduced in said cells by said

agent. Such cells may be, for example, isolated HSCs plated on to a plastic tissue culture plate and exhibiting SOX9 expression and collagen production.

Detailed description An anti-SOX9 agent for use in reducing expression of SOX9 in accordance with the invention may act to reduce SOX9 expression directly either at the level of SOX9 gene activation or by otherwise directly affecting SOX9 expression. The agent, for example, may be an siRNA, antisense-oligonucleotide or antisense peptide nucleic acid (PNA) which targets directly SOX9 expression. Such an agent may be expressed from a vector or provided as a conjugate and /or packaged for targeting the tissue of concern.

Thus, for treatment of liver fibrosis, desirably the agent will be packaged and /or conjugated to a second entity for targeting hepatocytes. For example, liver-targeting liposomes may be employed, e.g. liver-targeting charge-reversible Smarticles® (Novsom AG) or other liposomal formulations as previously proposed for delivery of siRNAs to liver cells (see e.g. www.alnylam.com). Recently, vitamin A linkage to liposomes has been proposed for specifically targeting siRNA to HSCs to treat liver cirrhosis but by targeting collagen chaperone heat shock protein 47 (Sato et al. (April 2008) Nat. Biotechnol. 26, 431-442). Various other approaches have been previously proposed for targeting oligonucleotide delivery, e.g, siRNAs, to specific cells such as antibody-mediated delivery (e.g. Song et al. (2005) Nature Biotech. 23^ 709-717) and ligand-targeted nanoparticles (e.g. Schifflers et al. (2004) Nucleic Acids Res. 32 149) and might be adapted for targeting SOX9 expression in the liver.

For treatment of a dermal fibrotic condition, e.g. skin keloid, a conjugate may be provided in which an antisense PNA, antisense oligonucleotide or siRNA is linked to an entity capable of promoting skin penetration and thereby suitable for topical delivery. Various compounds for this purpose have previously been described, e.g. a poly- pseudolysine (PPL), preferably for example, a 7 mer poly-pseudolysine (Peretto et al. (2003) Chem. Commun. 2312-2313; Kumar et al. (2006) Br. J. Dermatol. 155, 237). For topical delivery of an siRNA, reference may also be made to known proposed topical delivery of sRNAi for various dermatological conditions including hair removal (www. Sirna.com). Various other methods have been previously described for facilitating drug delivery through the skin barrier and are of interest in connection with treatment of dermal fibrotic conditions including, for example, ballistic-delivery of coated particles as well-known, for example, for DNA vaccine delivery, iontophoretic

methods, transdermal elecktrokinetic drug delivery (www. transportpharma.com), enhanced hydroalcoholic gel (EHG™) technology (www.ascendtherapeutics.com) and lipid-based delivery systems such as penetration enhancers (www.acrux.com), liposomes and liposome -like vesicles such as Transfersome® vesicles (www.idea- ag.de).

In a further aspect, the invention provides a pharmaceutical composition comprising an agent as described above for targeting SOX9 expression together with a pharmaceutically acceptable carrier or diluent. Such a composition may, for example, be a cream, lotion, ointment or gel for topical application to treat a skin condition such as skin keloid. It may be a composition for systemic delivery, e.g. comprising a liver- targeting vector.

In screening for agents for use in accordance with the invention, any cultured cells may be employed which exhibit detectable SOX9 expression or reporter gene activation from a SOX9 gene regulatory element-reporter gene construct, which may, for example, include a complete SOX9 promoter region or a portion or portions thereof which retain SOX9 promoter activity. Such reporter gene constructs, e.g. a SOX9 gene promoter-luciferase gene construct, will be introduced into cells by transfection and the cells thus transfected treated to activate reporter gene expression, e.g. by treatment to inhibit histone deacetylase activity as further discussed below. Preferably, SOX9- expressing cells chosen for screening as above will respond to SOX9 expression by production of type 1 and /or type 2 collagen; for example, fetal hepatocytes treated with Trichostatin A (TSA), an inhibitor of Class I and Class Il histone deacetylases, to cause activation of NF-Y, which will in turn activate the SOX9 promoter resulting in the expression of SOX9 protein and, consequently, type 2 collagen (see also the discussion in the exemplification of fetal hepatocytes and the discussion of HeLa cells transfected with reporter gene constructs and so treated). As a further example, as indicated above, the cells may preferably be HSCs plated on to a plastic tissue culture plate and exhibiting SOX9 expression and collagen production. For example, SOX9 expression may be detected by antibody detection or detection of encoding mRNA. Alternatively, or additionally, the level of type 1 and / or type 2 collagen may be determined.

The following exemplification illustrates the invention with reference to the following figures.

Brief description of the figures

Figure 1 : Luciferase activity in PANC1 cells transfected with S0X9 promoter region- luciferase gene constructs and treated with TSA or FK228. Lengths of 5' flanking region and/or mutations are shown to the left with corresponding fold induction of luciferase activity induced by TSA or FK228 shown to the right (mean from at least 3 separate experiments with error bars showing standard error).

Figure 2: Luciferase activity in HeLa cells transfected with S0X9 promoter region- luciferase gene constructs and treated with TSA. Again, lengths of 5' flanking region and/or mutations are shown to the left with corresponding fold induction of luciferase activity induced by TSA shown to the right (mean from at least 3 separate experiments with error bars showing standard error).

Figure 3: Quantified induction of COL2a by Western blotting in human fetal hepatocytes incubated with TSA. Fold induction has been normalised to β-actin and was standardised in each experiment against control cells incubated without TSA. Five separate experiments were carried out with line bars showing the standard error of the mean.

Figure 4: Effect of RNAi for SOX9 in human fetal hepatocytes expressing type 2 collagen. Expression, quantified by immunoblotting, was normalised to β-actin and standardised against control cells transfected with scrambled siRNA. * P < 0.001 compared to corresponding controls. Again, five separate experiments were carried out with line bars showing the standard error of the mean

Figure 5: Expression of SOX9, COLI a and COL2a by Western blotting in quiescent HSCs and HSCs after 7 and 10 days on plastic tissue culture plates. Fold induction was normalised to β-actin and standardised in each experiment agsinst protein levels in quiescent cells. Error bars represent the standard error of induction from 3 separate

HSC preparations.

Figure 6: TGF-β induction of SOX9 expression and SOX9 regulation of COL1 expression in HSCs.

A. Quantified induction of SOX9 expression by immunoblotting of HSCs and LX2 cells.

Fold induction in response to TGF-β (2ng/ml) compared to in its absence was analysed in 0% or 0.5% serum conditions. The fold induction was normalised to β-actin expression. Line bars show the standard error of induction from 3 separate HSC preparations or 3 separate experiments with LX2 cells.

B: siRNA abrogation of COLI a and COL2a in HSCs expressing SOX9. Expression by quantified Western blotting is shown relative to control (scrambled) oligonucleotides.

Line bars show the standard error of the mean form 3 separate HSC preparations.

* P<0.05 and " P<0.01 compared to the relevant controls.

Examples

Materials and methods Reagents and constructs

Trichostatin A (TSA) was purchased from Sigma (Sigma-Aldrich Ltd., UK); TGF-β was purchased from R & D Systems Europe Ltd., UK and FK228 was a gift from Professor Graham Packham (Cancer Sciences, University of Southampton). All other chemicals were purchased from Sigma or Thermo Fisher Scientific, UK. Promoter constructs were cloned into pGL3 Basic (Promega, UK) and numbered relative to the transcriptional start site. Mutations within the SOX9 p-192 promoter construct were generated by PCR with the oligonucleotides shown in Table 1.

Immortalized and primary cell culture PANC-1 and HeLa cell lines were purchased from the European Collection of Cell Cultures (ECACC). Human LX2 cells were a gift from Dr Scott Friedman (Mount Sinai School of Medicine, NY). Human fetal material was collected from first trimester termination of pregnancy with informed consent and following ethical approval from the Southampton & South West Hampshire Local Research Ethics Committee under guidelines issued by the Polkinghorne committee. Human fetal hepatocytes were prepared by mechanical disaggregation. Cells, other than rat primary hepatic stellate cells, were grown in monolayer at 5% CO 2 and 37°C in DMEM + L-Glutamine containing antibiotics supplemented with 10% fetal bovine serum (FBS) ('complete media'; PAA laboratories, Somerset, UK). Primary rat hepatic stellate cells were grown in DMEM containing 16% FBS. For TSA stimulation and /or transient transfection using Transfast (Promega Ltd) or gene silencing using Hiperfect (Qiagen Ltd), all cell types were plated at -60-70% density the day prior to treatment. For TUNEL assay (according to the manufacturer's instructions; Chemicon Ltd), activated (post-day 10) HSCs were cultured for 24 hrs in serum-free media following siRNA transfection. For immunocytochemistry, cells were plated onto glass slides coated with fibronectin (5 μg /cm 2 ; Sigma, UK). Where relevant, cells were stimulated with 25 ng/ml TSA in complete media for pulses of 8 hrs on 2 consecutive days. Immediately following the last pulse, cells were washed twice in PBS, and fixed in 4% paraformaldehyde (PFA). To assess the effect of TGF-β on SOX-9 expression, activated HSCs and LX2 cells were cultured

for 48 and 24 hrs respectively in 0% and 0.5% serum. TGF-β was then added for the next 24 hrs under the same low serum conditions.

CCU liver injury model and the preparation of rat hepatic stellate cells Liver fibrosis was induced by 5-week treatment of adult male Sprague-Dawley rats with carbon tetrachloride (CCI 4 ). This methodology along with tissue fixation and processing and the preparation of primary rat HSCs has been described previously (Smart et al., (2006) Hepatoloqy 44,1432-1440). Quiescent HSCs were isolated from the livers of untreated rats and plated onto tissue culture plastic as described previously (Smart et al. (2006) ibid). In this in vitro setting, HSCs replicate in vivo activation over the course of a week or so, as indicated above becoming positive for α-smooth muscle actin and adopting a profibrotic phenotype secreting COL1.

Gene expression analyses Antibodies which were used are listed in Table 2. Tissue preparation, immunohistochemistry, immunofluorescence and western blotting were performed as described previously (Piper et al. (2004) J. Endocrinol. 181. 1 1-23; Hearn et al. (2005) Diabetes 54, 1581-1587). Quantification of the protein band intensity after chemiluminescent detection of the immunoblotting was conducted using Quantity One Software (Bio-Rad Ltd, UK). For RT-PCR, RNA was extracted using Tri-Reagent (Sigma) and cDNA synthesized with Superscript III (Invitrogen) (Turnpenny et al. (2003) Stem cells 21, 598-609). PCR was carried out using 2 μl of cDNA with intron- spanning primers (see Table 3).

Luciferase, electrophoretic mobility shift and chromatin immunoprecipitation assays

Methodology for the luciferase assays and electrophoretic mobility shift assay (EMSA) has been described previously (Hanley et al. (2001 ) MoI. Endcrinol. 15, 57-68). Mutations of the proximal (p) and distal (d) CCAAT elements are shown in Table 1. Chromatin immunoprecipitation (ChIP) studies were carried out according to the manufacturer's instructions (Active Motif, Rixensart, Belgium) followed by PCR amplification of the SOX9 proximal promoter from -163 bas pairs to the transcriptional start site.

RNA interference In human fetal hepatocytes, SOX9 expression was silenced using commercial RNA oligonucleotides according to the manufacturer's instructions (Qiagen Ltd), which for

TUNEL were purchased pre-labelled with Alexa Fluor 555. For the primary rat HSCs,

RNA oligonucleotides were introduced into cells via Nucleofection (Amaxa Biosystems GmBh, Cologne, Germany) (Mazzocca et al. (2005) J. Biol. Chem. 280, 1 1329- 1 1339). Briefly, 2.5 x 10 6 HSC were resuspended in 100μl T-solution and combined with 2μl of 20μM siRNA. Cells were transfected using the U-25 pulsing parameter and immediately transferred to tubes containing 37°C pre-warmed culture media. Cells were cultured for 48 hrs on 12-well plates (protein extraction) or fibronectin-coated chamber slides (TUNEL assay).

Statistical analysis

Statistical analysis was carried out using one-way analysis of variance (ANOVA) with a Newman-Keuls post-hoc test or two-tailed Student T-test.

Preliminary studies with pancreatic ductal cell line Preliminary studies were carried out with the human pancreatic ductal cell line PANC-1 with a view to determining what might determine expression of SOX9 in human pancreatic progenitors but not in epithelial hepatocytes of developing human liver. The PANC-1 cell line has been identified previously to mimic fetal progenitors (Hardikar et al. (2003) Proc. Natl. Acad. Sci. USA 100, 7117-7122) and accordingly expresses SOX9. It was therefore chosen to first investigate regulation of the SOX9 gene promoter region in such cells. The SOX9 gene promoter is known to contain a site for cAMP response element binding protein (CREB) and two conserved CCAAT elements (Colter et al. (2005) Matrix Biol. 24, 185-197; Piera-Velazquez et al. (2007) Exp. Cell Res. 313, 1069-1079). The latter motifs are bound in vitro and in vivo by Nuclear Factor-Y. Histone deacetylases (HDACs) are capable of repressing gene expression by interaction with CCAAT-bound NF-Y, whereas HDAC- inhibitors relieve this repressed state of the basal promoter to favour gene expression (Imbriano et al. (2005) MoI. Cell. Biol. 25, 3737-3751 ). In PANC-1 cells, SOX9 promoter-luciferase reporter gene constructs containing at least one NF-Y/CCAAT site were found to be activated about 15-20 fold by TSA, an inhibitor of Class I and Class Il HDACs. The effect was replicated with the Class l-specific compound FK228 (also known as depsipeptide) and abolished by loss of functional CCAAT elements via either sequential truncation of the 5' flanking region or discrete mutations within a larger construct (see Figure 1 ). This finding of activation of the SOX9 promoter via CCAAT elements and ubiquitous NF-Y led to questioning of whether SOX9 could be induced in cells that do not normally express it.

Reporter gene expression in HeLa cells

In cultured HeLa cells transfected with S0X9 promoter-luciferase reporter gene constructs, TSA was again found to induce S0X9 promoter activity (about 150 to180- fold) both CCAAT elements were present. In their absence (by mutating both sites within the context of the p-192 construct (mut dpCCAAT)), TSA lost the vast majority of its ability to induce reporter gene expression (see Figure 2). In line with these findings, chromatin immunopreciptation (ChIP) after TSA treatment of HeLa cells demonstrated recruitment of transcription factor NB (TFIIB) at the proximal S0X9 promoter. TFIIB is critical for the assembly of multiple factors including RNA polymerase Il for the initiation of gene expression. Although cellular levels of NF-Y were unaltered, TSA increased NF-Y associated with the endogenous S0X9 promoter. These events at the proximal promoter were mirrored by a dose- and time- dependent accumulation of SOX9 protein in TSA-treated HeLa cells.

Expression of SOX9 in human fetal hepatocytes

Primary cultures of human early fetal heptocytes were established. TSA-induction of SOX9 expression was again found to be dose-responsive as determined by Western blotting of SOX9. Maximal stmulation by TSA occurred at 25 ng/ml. Neither pancreatic progenitor nor neuroprogenitor cell markers were detected following SOX9 up- regulation by TSA treatment. However, by RT-PCR 'chondrogenic' ECM markers were induced; COL2A1 and COMP, both regulated by SOX9 in chondrocytes, were induced. The increase in COL2A1 transcripts was mirrored by dose-dependent increases of type 2 collagen deposition. At 25 ng/ml TSA, when SOX9 was maximally stimulated, COL2 was increased approximately 5-fold consistent with known transcriptional regulation of COL2A1 by SOX9 (Bell et al.(1997) Nat. Genetics 16, 174); see Figure 3. Type 2 collagen expression was decreased by abrogating SOX9 levels using RNAi (see Figure 4). Similar decreases were observed using oligonucleotides targeted to one other region of the SOX9 transcript.

The genes encoding both collagen oligomeric protein (COMP) and type 1 collagen (COL1A2) have both recently been described as potential SOX9 targets (Ylostalo et al. ibid). COMP was found to be increased by RT-PCR; but different isoforms made it difficult to assess COMP protein levels reliably. Neither COL1A2 transcripts nor COL1 protein were significantly altered following induction of SOX9 by TSA. This might be explained by evidence that TSA elicits a broad alteration of gene expression including

a direct negative effect on COL1A2 expression (Rombouts et al. (2002) Exp. Cell Res.

278, 184-197; Niki et al. (1999) Hepatology 29, 858-867).

SOX9 involvement in in vivo and in vitro models of liver fibrosis CCI 4 injections were used to induce liver fibrosis in rats as noted above with deposition of types 1 and 2 collagen being found in fibrotic livers. By immunochemistry, nuclear SOX9 was also detected within fibrotic regions.

Quiesent HSCs were also isolated from rat livers and plated on to tissue culture plastic in serum where they became activated over the course of a week or so and adopted the myofibroblast-like appearance. Nuclear SOX9 was present in cells with cytoplasmic αSMA. SOX9 protein was increased 7 days after plating; more so at day 10, alongside the detection of type 1 and type 2 collagen (see Figure 5).

To additionally study the effect of TGF-β on SOX9 expression, activated HSCs were placed in low serum conditions as noted above. TGF-β increased SOX9 expression approximately 3-fold. As noted hereinbefore, under similar conditions, SOX9 was also induced in cells of the cell line LX2, a human model of HSCs (see Figure 6A).

This data fits with existing knowledge of signalling mechanisms in fibrosis. TGF-β signalling is a major influence in promoting HSC activation and subsequent COL1 expression; FGF2 augments ECM deposition (Iredale (2007) ibid; Henderson and lredale (2007) Clin. Sci. V\2, 265-280; Yu et al. (2003) Am. J. Pathol. 163,1653-1662). Similar observation of association of TGF-β with SOX9 expression was previously observed in developing chrondrocytes (Kawakami et al (2006) Curr. Opin Cell Biol. 1|5, 723-729). FGF2 has been reported to increase both SOX9 transcripts and protein in a chondrosarcoma cell line (Schaefer et al. (2003) Osteoarthritis Cartilage V\_, 233-241 ). Moreover, both TGF-β and FGF-2 promote epithelial-to-meschymal transistion (EMT) whereby quiescent epithelial cells morph into myofibroblast cells charcaterised by α- SMA. In development, this process induces SOX9 expression during the diiferentiation of chrondrocytes and astrocytes (Kalluri and Neilson (2003) Nat. Med. 13, 952-961 ). However , EMT is also a forerunner of organ fibrosis when the resultant mesenchymal cells express abundant COL1 as part of the fibrotic matrix. Recent data have provided evident of EMT as part of renal, hepatic and cardiac fibrosis (Henderson and Iredale ibid; Zeisberg et al. (2007) Nat. Med. 13, 952-961 ; Zeisberg et al. J. Biol. Chem. 282, 23337-23347). Although the origin of HSCs remains unclear, the finding of the

inventors that activated HSCs are marked by all of α-SMA expression, COL1 deposition and expression of SOX9 provides striking similarity with previously recognised post-EMT mesenchymal cells.

Furthermore, as indicated above, in activated rat HSCs, SOX9 has now been shown to be abrogated by RNAi. Following reduction of SOX9 protein levels by approximately 60%, COL1 was lowered by a similar magnitude, confirming a direct role for SOX9 in COL1 expression in activated HSCs. COL2 in activated HSCs was also abrogated by lower levels of SOX9 (see Figure 6B). Similar results were obtained by targeting one other region of the SOX9 transcript.

These findings demonstrate a newly discovered molecular pathway for fibrosis in the liver that seems applicable to other organs involving inappropriate expression of SOX9, a transcription factor that is otherwise central to normal human development. The finding of restriction of SOX9 to the disease-causing activated cell type rather than its quiescent counterpart, supports SOX9 abrogation as an attractive prospect for halting progress of liver fibrosis towards cirrhosis.

Table 1. Oligonucleotides used to mutate sites within the SOX9 p-192 promoter construct.

Gene Forward primer Reverse primer

δdCCAAT TCGGGCATGATCAGCTGCCT GCTGATCATGCCCGATTTTG

(TCCA) (SEQ ID NO. 1 ) (TGGA) (SEQ ID NO. 2)

δpCCAAT GCTGTGCGGGTGGCTCT CACCCGCACAGCACAGC

(ATTG) (SEQ ID NO. 3) (CAAT) (SEQ ID NO. 4)

Mutations are in bold with wildtype sequence for each oligonucleotide shown below in parentheses.

The SEQ ID NOs refer to the mutants. The wildtype sequences corresponding to SEQ ID NOs 1-4 are provided in ^

SEQ ID NOs 25-28, respectively.

Table 2. Antibodies used in this study. [Supplementary reference: Fisher et al. (1995) Acta Orthop. Scand. 66, 61-65: Antisera and cDNA probes to human and certain animal model bone matrix non-collagenous proteins]

Table 3 RT-PCR primers and conditions

Product

Gene Forward primer Reverse primer (bp)

C0L1 acctccggctcctgctcctcttag (SEQ ID NO. 5) ccctcgacgccggtggtttcttg (SEQ ID NO. 6) 319

C0L2a ttcagctatggagatgacaatc (SEQ ID NO. 7) agagtcctagagtgactgag (SEQ ID NO. 8) 472

C0L3 ggcccacctggtcctgtcg (SEQ ID NO. 9) atctccataatacggggcaaaacc (SEQ ID NO. 519

10)

C0L4 cctgccgggcctactggt (SEQ ID NO. 11 ) ggggcacggtgggatctgaatggt (SEQ ID NO. 480

12) COMP cggcaacgggatcctctgtggtc (SEQ ID NO. cgcctgatccgggttgctcttct (SEQ ID NO. 14) 478

13) PDX1 ggatgaagtctaccaaagctcacgc (SEQ ID NO. ccagatcttgatgtgtctctcggtc (SEQ ID NO. 16) Lee

15) ISL1 cgtgcccgctccaaggtgtatca (SEQ ID NO. cattgggctgctgctgctggagtt (SEQ ID NO. 18) 478

17)

HLXB9 ON gcacccggcgctctcctactcgt (SEQ ID NO. 19) ccgccgccgcccttctgtttctc (SEQ ID NO. 20) 420

S0X2 ggcacccctggcatggctcttg (SEQ ID NO. 21 ) ttcttgtcggcatcgcggtttttg (SEQ ID NO. 22) 484

HPRT cctggcgtcgtgattagtgatgat (SEQ ID NO. agcttgcgaccttgacca (SEQ ID NO. 24) 454

23)

SOX2 primers are not intron-spanning.




 
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