James Pritchett

Understanding the role of SOX9 in acquired diseases: lessons from development.

The transcription factor SOX9 is crucial for multiple aspects of development. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder concordant with the expression profile of SOX9 during embryogenesis. The mechanistic understanding of development has revealed roles for SOX9 in regulating cartilage extracellular matrix (ECM) production and cell proliferation, among others. More recently, it transpires that SOX9 becomes expressed and induces destructive ECM components in organ fibrosis and related disorders. Although commonly absent from the parent cell type, SOX9 is expressed in a wide range of cancers, where it regulates cell proliferation. These data have potential diagnostic, prognostic and therapeutic relevance, suggesting that disease mechanisms might result from re-expressing this developmental transcription factor in ectopic locations.

Osteopontin is a novel downstream target of SOX9 with diagnostic implications for progression of liver fibrosis in humans

Osteopontin (OPN) is an important component of the extracellular matrix (ECM), which promotes liver fibrosis and has been described as a biomarker for its severity. Previously, we have demonstrated that Sex‐determining region Y‐box 9 (SOX9) is ectopically expressed during activation of hepatic stellate cells (HSC) when it is responsible for the production of type 1 collagen, which causes scar formation in liver fibrosis. Here, we demonstrate that SOX9 regulates OPN. During normal development and in the mature liver, SOX9 and OPN are coexpressed in the biliary duct. In rodent and human models of fibrosis, both proteins were increased and colocalized to fibrotic regions in vivo and in culture‐activated HSCs. SOX9 bound a conserved upstream region of the OPN gene, and abrogation of Sox9 in HSCs significantly decreased OPN production. Hedgehog (Hh) signaling has previously been shown to regulate OPN expression directly by glioblastoma (GLI) 1. Our data indicate that in models of liver fibrosis, Hh signaling more likely acts through SOX9 to modulate OPN. In contrast to Gli2 and Gli3, Gli1 is sparse in HSCs and is not increased upon activation. Furthermore, reduction of GLI2, but not GLI3, decreased the expression of both SOX9 and OPN, whereas overexpressing SOX9 or constitutively active GLI2 could rescue the antagonistic effects of cyclopamine on OPN expression. Conclusion: These data reinforce SOX9, downstream of Hh signaling, as a core factor mediating the expression of ECM components involved in liver fibrosis. Understanding the role and regulation of SOX9 during liver fibrosis will provide insight into its potential modulation as an antifibrotic therapy or as a means of identifying potential ECM targets, similar to OPN, as biomarkers of fibrosis. (HEPATOLOGY 2012;56:1108–1116)

Stromal-derived factor 1 signalling regulates radial and tangential migration in the developing cerebral cortex

Stromal-derived factor 1 (SDF-1), a known chemoattractant, and its receptor CXCR4 are widely expressed in the developing and adult cerebral cortex. Recent studies have highlighted potential roles for SDF-1 during early cortical development. In view of the current findings, our histological analysis has revealed a distinct pattern of SDF-1 expression in the developing cerebral cortex at a time when cell proliferation and migration are at peak. To determine the role of chemokine signalling during early cortical development, embryonic rat brain slices were exposed to a medium containing secreted SDF-1 to perturb the endogenous levels of chemokine. Alternatively, brain slices were treated with 40 µM of T140 or AMD3100, known antagonists of CXCR4. Using these experimental approaches, we demonstrate that chemokine signalling is imperative for the maintenance of the early cortical plate. In addition, we provide evidence that both neurogenesis and radial migration are concomitantly regulated by this signalling system. Conversely, interneurons, although not dependent on SDF-1 signalling to transgress the telencephalic boundary, require the chemokine to maintain their tangential migration. Collectively, our results demonstrate that SDF-1 with its distinct pattern of expression is essential and uniquely positioned to regulate key developmental events that underlie the formation of the cerebral cortex.

PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis.

Stromal derived factor-1 exerts differential regulation on distinct cortical cell populations in vitro

BackgroundStromal derived factor (SDF-1), an alpha chemokine, is a widely known chemoattractant in the immune system. A growing body of evidence now suggests multiple regulatory roles for SDF-1 in the developing nervous system.ResultsTo investigate the role of SDF-1 signaling in the growth and differentiation of cortical cells, we performed numerous in vitro experiments, including gene chip and quantitative RT-PCR analysis. Using SDF-1 medium and AMD3100, a receptor antagonist, we demonstrate that the chemokine signaling regulates key events during early cortical development. First, SDF-1 signaling maintains cortical progenitors in proliferation, possibly through a mechanism involving connexin 43 mediated intercellular coupling. Second, SDF-1 signaling upregulates the differentiation of cortical GABAergic neurons, independent of sonic signaling pathway. Third, SDF-1 enables the elongation and branching of axons of cortical glutamatergic neurons. Finally, cortical cultures derived from CXCR4-/- mutants show a close parallel to AMD3100 treatment with reduced cell proliferation and differentiation of GABAergic neurons.ConclusionResults from this study show that SDF-1 regulates distinct cortical cell populations in vitro.

Epimorphin Alters the Inhibitory Effects of SOX9 on Mmp13 in Activated Hepatic Stellate Cells

Background and Aims Liver fibrosis is a major cause of morbidity and mortality. It is characterised by excessive extracellular matrix (ECM) deposition from activated hepatic stellate cells (HSCs). Although potentially reversible, treatment remains limited. Understanding how ECM influences the pathogenesis of the disease may provide insight into novel therapeutic targets for the disease. The extracellular protein Epimorphin (EPIM) has been implicated in tissue repair mechanisms in several tissues, partially, through its ability to manipulate proteases. In this study, we have identified that EPIM modulates the ECM environment produced by activated hepatic stellate cells (HSCs), in part, through down-regulation of pro-fibrotic Sex-determining region Y-box 9 (SOX9). Methods Influence of EPIM on ECM was investigated in cultured primary rat HSCs. Activated HSCs were treated with recombinant EPIM or SOX9 siRNA. Core fibrotic factors were evaluated by immunoblotting, qPCR and chromatin immunoprecipitation (ChIP). Results During HSC activation EPIM became significantly decreased in contrast to pro-fibrotic markers SOX9, Collagen type 1 (COL1), and α- Smooth muscle actin (α-SMA). Treatment of activated HSCs with recombinant EPIM caused a reduction in α-SMA, SOX9, COL1 and Osteopontin (OPN), while increasing expression of the collagenase matrix metalloproteinase 13 (MMP13). Sox9 abrogation in activated HSCs increased EPIM and MMP13 expression. Conclusion These data provide evidence for EPIM and SOX9 functioning by mutual negative feedback to regulate attributes of the quiescent or activated state of HSCs. Further understanding of EPIMs role may lead to opportunities to modulate SOX9 as a therapeutic avenue for liver fibrosis.

SOX9 predicts progression toward cirrhosis in patients while its loss protects against liver fibrosis

Fibrosis and organ failure is a common endpoint for many chronic liver diseases. Much is known about the upstream inflammatory mechanisms provoking fibrosis and downstream potential for tissue remodeling. However, less is known about the transcriptional regulation in vivo governing fibrotic matrix deposition by liver myofibroblasts. This gap in understanding has hampered molecular predictions of disease severity and clinical progression and restricted targets for antifibrotic drug development. In this study, we show the prevalence of SOX9 in biopsies from patients with chronic liver disease correlated with fibrosis severity and accurately predicted disease progression toward cirrhosis. Inactivation of Sox9 in mice protected against both parenchymal and biliary fibrosis, and improved liver function and ameliorated chronic inflammation. SOX9 was downstream of mechanosignaling factor, YAP1. These data demonstrate a role for SOX9 in liver fibrosis and open the way for the transcription factor and its dependent pathways as new diagnostic, prognostic, and therapeutic targets in patients with liver fibrosis.

Zeolite-embedded silver extends antimicrobial activity of dental acrylics

The insertion of prosthetic devices into the oral cavity affects the oral microflora and results in accumulation of microorganisms on the prosthetic surface. Such fouling of denture surfaces can lead to a number of oral diseases and consequently to the replacement of the denture. Here, we report the post-synthesis introduction of silver in zeolite-loaded dental acrylic (DAZ) resins that does not influence the mechanical or aesthetic properties of the DA resins, and provides them with a long-term antimicrobial activity. Na-FAU zeolite (2 wt%) was incorporated into DA resin, which was conventionally processed and cut into 10 mm × 20 mm × 3 mm coupons. The Na+ in the zeolite was then exchanged with Ag+ via immersion of the DAZ coupons in 0.01 M AgNO3 solution to obtain DAZ/Ag-treated coupons used in antimicrobial tests. Antimicrobial tests showed that the DAZ/Ag-treated coupons were active against Candida albicans (a reference and a clinically relevant strain), Streptococcus mutans and Fusobacterium nucleatum. Ag leaching tests on the Ag-charged coupons at 1, 2, 3, 4, 7, 14, 30 and 45 days of incubation in distilled water at 37 °C, indicated sustained release of silver. Antimicrobial tests using a reference Candida albicans strain showed that the leached coupons retained antimicrobial activity after 45 days immersion in distilled water, but, after 60 days incubation no antimicrobial activity was observed. Cytotoxicity assay results indicated that the DAZ/Ag-treated coupons showed no additional cytotoxicity compared to neat dental acrylic coupons.

OC-028 Identification of SOX9 as a novel mechanism to explain aspects of liver fibrosis

Introduction Fibrosis of the liver is characterised by progressive accumulation of extracellular matrix (ECM) proteins and is a major cause of morbidity and mortality in the UK. Several cell-types are responsible for this, but a major role is played by activation of the hepatic stellate cell (HSC). We have identified ectopic expression of the Sry-box transcription factor, SOX9, in activated HSCs as a novel mechanism to explain aspects of liver fibrosis. Methods Livers were collected from carbon tetrachloride induced fibrotic and control rats and processed for fixed tissue. Rat hepatic stellate cells (HSCs) were isolated using established perfusion techniques and cultured on tissue culture plastic to activate over 4, 7 and 10 days. The immortalised human LX2 cell line was also used. Results In fixed fibrotic rat livers α-smooth muscle actin (α-sma) was detected in activated HSCs with collagen type 1 (Col1) rich fibrotic tracts found disrupting normal tissue architecture. Nuclear Sox9 staining was detected in the same regions as α-sma and Col1. In quiescent HSCs isolated from rat liver, negative for α-sma and col1, Sox9 was absent. However during activation of HSCs, both Sox9 and Col1 were robustly expressed. In these activated Col1-expressing HSCs, Sox9 localised to the nucleus surrounded by α-sma positive cytoplasm. SOX9 knockdown in activated HSCs using RNA interference caused a commensurate reduction in Col1 protein expression (∼60%). The pro-fibrotic cytokine transforming growth factor β (TGF-β) induced expression of SOX9 by ∼3-fold in rat HSCs and ∼2.5-fold in the human stellate cell line LX2s. Conclusion We have identified SOX9 as a novel mediator of ECM in liver fibrosis. Our data demonstrate that SOX9 expression occurs during activation of HSCs, a major cell type responsible for liver fibrosis, when, under the influence of TGF-β signalling it causes Col1 production, the predominant collagen deposited in organ fibrosis. Lessening SOX9 levels similarly reduced Col1 production. These data suggest targeted reduction of SOX9 offers potential therapeutic application to ameliorate fibrosis and related conditions of the liver.

Sdf-1/Cxcr4 signalling regulates proliferation during cortical development

rescued in Shh;Gli3 double mutants. We reasoned that, if Foxg1 exerts its effects on ventral telencephalon through participation in Shh-mediated antagonism of Gli3 activity, then ventral defects in Foxg1 mutants should be rescued when Gli3 activity is removed. We therefore generated Foxg1;Gli3 double mutant embryos. These showed a partial rescue of the Foxg1 mutant phenotype, in that expression of several ventral markers was restored. This suggests that Foxg1 does indeed participate in Shh-mediated antagonism of Gli3 activity. However, Foxg1;Gli3 telencephalon still exhibits several D/V patterning defects, consistent with further, Hh-independent, roles for Foxg1 in telencephalic patterning. These findings show that Foxg1 plays a central and multi-functional role in the development of the ventral telencephalon and its derivatives.