Hepatocellular carcinoma (HCC) commonly arises in metabolic dysfunction-associated steatohepatitis (MASH), alcohol-related liver disease (ALD), and metabolic dysfunction-associated ALD (MetALD), yet how zonal metabolic programs govern tumor lineage and immune responses remains unclear. Here, using complementary murine models of steatohepatitis-associated hepatocarcinogenesis, we show that CTNNB1-mutant MASH-HCC originates from periportal and midlobular hepatocytes through perivenous reprogramming. This transition is characterized by β-catenin activation, loss of periportal metabolic functions, and induction of the immunosuppressive IDO1-kynurenine-AhR axis. In contrast, ethanol exposure suppresses perivenous xenobiotic programs, destabilizes the β-catenin/AhR/CAR axis, and increases tumor heterogeneity by generating both progenitor/biliary- and hepatocyte-derived MetALD-HCC that remain sensitive to anti-programmed death-1 (aPD1) therapy. Pharmacologic AhR inhibition or hepatocyte-specific β-catenin deletion reduces MASH-HCC burden and restores sensitivity to aPD1 treatment. Together, these findings identify AhR as a central mediator of β-catenin-driven tumor immunosuppression and a potential therapeutic target in CTNNB1-mutant HCC, highlighting context-dependent mechanisms of immune escape in alcohol-associated HCC. Alcohol consumption and high fat diet can drive liver cancer through distinct pathways. Here, the authors characterize three murine models of steatohepatitis-associated hepatocarcinogenesis that recapitulate metabolic dysfunction-associated steatohepatitis (MASH), alcohol-related liver disease (ALD), and their overlapped condition, MetALD, showing that alcohol reshapes liver zonal plasticity and β-catenin-AhR signaling to alter tumor origin and increase immunotherapy sensitivity.

Background: Extracellular matrix protein 1 (ECM1) can inhibit TGFβ activation, but its antifibrotic action remains largely unknown. This study aims to investigate ECM1 function and its physical interaction with the profibrotic connective tissue growth factor (CTGF) in fibrosis and ductular reaction (DR). Methods: Ecm1 knockouts or animals that ectopically expressed this gene were subjected to induction of liver fibrosis and DR by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or α-naphthyl-isothiocyanate (ANIT). ECM1 and CTGF were also examined in the livers of patients with alcohol-associated liver disease (ALD) or ethanol-exposed animals that were fed the western diet for 4 months in the WDA model with liver pathology resembing ALD in patients. Results: ECM1 bound to CTGF in yeast two-hybrid systems, cultured liver cells, and cholestatic livers damaged by DDC or α-naphthyl-isothiocyanate. This interaction blocked integrin αvβ6-mediated TGFβ activation, thereby reducing fibrotic responses in vitro. ECM1 downregulation was associated with biliary CTGF induction during human ALD progression. In experimental models, Ecm1 loss enhanced susceptibility to DDC-induced cholestasis with upregulation of Ctgf, αvβ6, alpha-smooth muscle actin, procollagen type I, serum transaminase, and total bilirubin levels in germline knockouts, whereas forced expression of this gene significantly attenuated DR and biliary fibrosis after the feeding of DDC or α-naphthyl-isothiocyanate containing diets. Moreover, ectopic Ecm1 inhibited not only alcohol-associated fibrosis but also TGFβ-mediated deregulation of hepatocyte nuclear factor 4α, preventing the production of the fetal p2 promoter-driven isoforms in the WDA model. Conclusions: We uncover a novel antifibrotic action by ECM1 that binds CTGF and inhibits integrin αvβ6-mediated TGFβ activation. Targeting its loss has therapeutic potential for the treatment of DR and liver fibrosis in chronic conditions, such as cholangiopathy and ALD.

Liver fibrosis is the common outcome of many chronic liver diseases, resulting from altered cell-cell and cell-matrix interactions that promote hepatic stellate cell (HSC) activation and excessive matrix production. This study aimed to investigate functions of cellular communication network factor 2 (CCN2)/Connective tissue growth factor (CTGF), an extracellular signaling modulator of the CYR61/CTGF/Nov (CCN) family, in liver fibrosis. Tamoxifen-inducible conditional knockouts in mice and hepatocyte-specific deletion of this gene in rats were generated using the Cre-lox system. These animals were subjected to peri-central hepatocyte damage caused by carbon tetrachloride. Potential crosstalk of this molecule with a new profibrotic pathway mediated by the Slit2 ligand and Roundabout (Robo) receptors was also examined. We found that Ccn2/Ctgf was highly upregulated in periportal hepatocytes during carbon tetrachloride-induced hepatocyte damage, liver fibrosis and cirrhosis in mice and rats. Overexpression of this molecule was observed in human hepatocellular carcinoma (HCC) that were surrounded with fibrotic cords. Deletion of the Ccn2/Ctgf gene significantly reduced expression of fibrosis-related genes including Slit2, a smooth muscle actin (SMA) and Collagen type I during carbon tetrachloride-induced liver fibrosis in mice and rats. In addition, Ccn2/Ctgf and its truncated mutant carrying the first three domains were able to interact with the 7th -9th epidermal growth factor (EGF) repeats and the C-terminal cysteine knot (CT) motif of Slit2 protein in cultured HSC and fibrotic murine livers. Ectopic expression of Ccn2/Ctgf protein upregulated Slit2, promoted HSC activation, and potentiated fibrotic responses following chronic intoxication by carbon tetrachloride. Moreover, Ccn2/Ctgf and Slit2 synergistically enhanced activation of phosphatidylinositol 3-kinase (PI3K) and AKT in primary HSC, whereas soluble Robo1-Fc chimera protein could inhibit these activities. These observations demonstrate conserved cross-species functions of Ccn2/Ctgf protein in rodent livers. This protein can be induced in hepatocytes and contribute to liver fibrosis. Its novel connection with the Slit2/Robo signaling may have therapeutic implications against fibrosis in chronic liver disease.

Yes-associated protein (YAP), a central effector in the Hippo pathway, is involved in the regulation of organ size, stem cell self-renewal, and tissue regeneration. In this study, we observed YAP activation in patients with alcoholic steatosis, hepatitis, and cirrhosis. Accumulation of this protein in the nucleus was also observed in murine livers that were damaged after chronic-plus-single binge or moderate ethanol ingestion combined with carbon tetrachloride intoxication (ethanol/CCl4). To understand the role of this transcriptional coactivator in alcohol-related liver injury, we knocked out the Yap1 gene in hepatocytes of floxed homozygotes through adeno-associated virus (AAV8)-mediated deletion utilizing Cre recombinase. Yap1 hepatocyte-specific knockouts (KO) exhibited hemorrhage, massive hepatic necrosis, enhanced oxidative stress, elevated hypoxia, and extensive infiltration of CD11b(+) inflammatory cells into hepatic microenvironments rich for connective tissue growth factor (Ctgf) during ethanol/CCl4-induced liver damage. Analysis of whole-genome transcriptomics indicated upregulation of genes involved in hypoxia and extracellular matrix (ECM) remodeling, whereas genes related to hepatocyte proliferation, progenitor cell activation, and ethanol detoxification were downregulated in the damaged livers of Yap1 KO. Acetaldehyde dehydrogenase (Aldh)1a1, a gene that encodes a detoxification enzyme for aldehyde substrates, was identified as a potential YAP target because this gene could be transcriptionally activated by a hyperactive YAP mutant. The ectopic expression of the human ALDH1A1 gene caused increase in hepatocyte proliferation and decrease in hepatic necrosis, oxidative stress, ECM remodeling, and inflammation during ethanol/CCl4-induced liver damage. Taken together, these observations indicated that YAP was crucial for liver repair during alcohol-associated injury. Its regulation of ALDH1A1 represents a new link in liver regeneration and detoxification.

During progression to type 1 diabetes, insulin-producing β-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered β-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.

The liver possesses an extraordinary ability to regenerate after injury. Hepatocyte-driven liver regeneration is the default pathway in response to mild-to-moderate acute liver damage. When replication of mature hepatocytes is blocked, facultative hepatic progenitor cells (HPCs), also referred to as oval cells (OCs) in rodents, are activated. HPC/OCs have the ability to proliferate clonogenically and differentiate into several lineages including hepatocytes and bile ductal epithelia. This is a conserved liver injury response that has been studied in many species ranging from mammals (rat, mouse, and human) to fish. In addition, improper HPC/OC activation is closely associated with fibrotic responses, characterized by myofibroblast activation and extracellular matrix production, in many chronic liver diseases. Matrix remodeling and metalloprotease activities play an important role in the regulation of HPC/OC proliferation and fibrosis progression. Thus, understanding molecular mechanisms underlying HPC/OC activation has therapeutic implications for rational design of anti-fibrotic therapies.

miRNAs are involved in Liver regeneration, and their expression is dysregulated in hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF), a direct target of miR-133b, is crucial in the ductular reaction (DR)/oval cell (OC) response for generating new hepatocyte lineages during liver injury in the context of hepatotoxin-inhibited hepatocyte proliferation. Herein, we investigate whether miR-133b regulation of CTGF influences HCC cell proliferation and migration, and DR/OC response. We analyzed miR-133b expression and found it to be down-regulated in HCC patient samples and induced in the rat DR/OC activation model of 2-acetylaminofluorene with partial hepatectomy. Furthermore, overexpression of miR-133b via adenoviral system in vitro Led to decreased CTGF expression and reduced proliferation and Transwell migration of both HepG2 HCC cells and WBF-344 rat OCs. In vivo, overexpression of miR-133b in DR/OC activation models of 2-acetylaminofluorene with partial hepatectomy in rats, and 3,5-diethoxycarbonyl-1,4-dihydrocollidine in mice, led to down-regulation of CTGF expression and OC proliferation. Collectively, these results show that miR-133b regulation of CTGF is a novel mechanism critical for the proliferation and migration of HCC cells and OC response.

Abstract Introduction: Obesity and type 2 diabetes are risk factors for liver cancer due to a transition from steatosis to nonalcoholic steatohepatitis (NASH), resulting in liver fibrosis. If left untreated, liver fibrosis can progress into cirrhosis leading to hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF) is a pro-fibrotic matricellular protein that is highly expressed during hepatocarcinogenesis. This study aims to investigate the involvement of CTGF in nonalcoholic steatohepatitis (NASH) and liver cancer development during diabetic conditions induced by streptozotocin (STZ) treatment and the feeding of a high fat diet (HFD) in mice. Methods: Transgenic male mice expressing green fluorescent protein (GFP) under the control of Ctgf promoter (Ctgfp-GFP), liver specific CTGF knockouts (CtgfΔhep/Δhep), and control mice that contained two alleles of floxed Ctgf (Ctgffloxed/floxed) were given STZ (200 ng/pup) at postnatal day 2 (P2) through subcutaneous injection and were fed HFD at postnatal week four to induce NASH and HCC. The treated animals were harvested at postnatal week 5, 12, and 20 for early steatosis, fibrosis and liver cancer development respectively. CTGF expression was analyzed by immunofluorescent staining, Western analysis, and qRT-PCR. In addition, the mouse liver cancer RT2 profiler PCR array was screened to identify cancer-related genes that were differentially expressed during NASH and HCC development comparing CtgfΔhep/Δhep and Ctgffloxed/floxed mice. Results: All three types of animals developed NASH and HCC in response to STZ and HFD feeding. Liver pathologies ranging from steatosis, NASH, liver fibrosis, and nodule formation, to intratumoral angiogenesis were observed as discrete molecular and histological stages in the STZ/HFD induced NASH-HCC model. Immunofluorescent analysis of Ctgfp-GFP reporter mice showed induction of the Ctgf gene in vascular endothelial cells, biliary epithelial cells, hepatocytes, and inflammatory cells in STZ/HFD livers. We utilized the mouse liver cancer RT2 profiler PCR array and compared the expression of 90 liver cancer related genes between CtgfΔhep/Δhep and Ctgff/f tumors that developed after 12-week HFD feeding. 12 upregulated genes and 10 downregulated genes were identified in Ctgfk/k mice compared to Ctgff/f animals. Conclusion: We successfully established a NASH-HCC model combining STZ and HFD treatment. CTGF expression was found in multiple cell types including endothelial cells, cholangiocytes, inflammatory cells, and hepatocytes. Liver specific deletion of CTGF was associated with differential expression of groups of genes involved in cell proliferation and apoptosis. The functional relationship between these genes and CTGF in liver cancer development in the setting of metabolic syndrome needs to be characterized in future studies. Citation Format: Liya Pi, Marda Jorgensen, Seh-Hoon Oh, Altin Gjymishka, Bryon E. Petersen. The involvement of connective tissue growth factor in nonalcoholic steatohepatitis and liver cancer development under diabetic condition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5107.

Obesity poses an increased risk of developing metabolic syndrome and closely associated nonalcoholic fatty liver disease, including liver cancer. Satiety hormone leptin-deficient (ob/ob) mice, considered paradigmatic of nutritional obesity, develop hepatic steatosis but are less prone to developing liver tumors. Sustained activation of peroxisome proliferator-activated receptor alpha (PPAR alpha) in ob/ob mouse liver increases fatty acid oxidation (FAO), which contributes to attenuation of obesity but enhances liver cancer risk. To further evaluate the role of PPAR alpha-regulated hepatic FAO and energy burning in the progression of fatty liver disease, we generated PPAR alpha-deficient ob/ob (PPAR alpha(Delta)ob/ob) mice. These mice become strikingly more obese compared to ob/ob littermates, with increased white and brown adipose tissue content and severe hepatic steatosis. Hepatic steatosis becomes more severe in fasted PPAR alpha(Delta)ob/ob mice as they fail to up-regulate FAO systems. PPAR alpha(Delta)ob/ob mice also do not respond to peroxisome proliferative and mitogenic effects of PPAR alpha agonist Wy-14,643. Although PPAR alpha(Delta)ob/ob mice are severely obese, there was no significant increase in liver tumor incidence, even when maintained on a diet containing Wy-14,643. We conclude that sustained PPAR alpha activation-related increase in FAO in fatty livers of obese ob/ob mice increases liver cancer risk, whereas deletion of PPAR alpha in ob/ob mice aggravates obesity and hepatic steatosis. However, it does not lead to liver tumor development because of reduction in FAO and energy burning.

This commentary will address the article published in the Journal of Clinical Investigation in December 2014 by Kordes et al.1 The article describes a method for isolating a subpopulation of hepatic stellate cells (HSCs) based on retinoid storage. The researchers isolated these cells from GFP+ rats and transplanted them into wild-type rats. Recipient rats were then subjected to one of two different models for liver injury (retrorsine/partial hepatectomy [PHx] and 2-acetylaminofluorene/PHx). Liver regeneration in each of these models is mediated primarily by liver progenitor cells, and in each case, GFP+ cells were found to contribute to restoration of the mesenchymal cell, progenitor cell, hepatocyte, and cholangiocyte populations. The data presented by Kordes et al. clearly establish the multipotency of hepatic retinoid storing cells within the liver. Ever since hepatic “oval” cells were first characterized by Dr. Farber in the 1950s, the field of liver progenitor cell research has attempted to define this cell population based on the expression of a discrete panel of markers, much as was done with hematopoietic stem cells.2 Liver regeneration is a very complex process that depends on a well-orchestrated series of signals that govern the repopulation and reorganization of damaged liver tissue. For the most part, hepatocytes are able to effectively restore any parenchymal volume lost to injury. However, in the case of chronic liver injury and in certain liver diseases, mature hepatocytes are unable to affect liver repair. In these scenarios, facultative liver stem cells are recruited to augment the regenerative response. Over the past four decades, a variety of strategies have been developed for the isolation of these liver progenitor cells. These strategies have included density gradient centrifugation, cell sorting based on membrane antigen expression (e.g., EpCAM and Thy-1), and cell panning to deplete unwanted cells. However, no consensus has yet been reached on a definitive strategy for the isolation of liver progenitor cells. Two key factors have complicated the universal acceptance of a single isolation technique. The first of these is that the progenitor cell population represents a spectrum of differentiation states, presenting an enormous challenge in the development of a unified isolation strategy. Second, it has become apparent that different types of injuries activate different progenitor cell populations. This creates a division among groups that favor various experimental injury models. An article by Kuwahara et al. describes four distinct microanatomical compartments from which asymmetrically dividing cells emerge in response to specific types of liver injury.3 The first location lies at the junction of the hepatocyte bile canaliculi and the proximal biliary ductules at the canals of Hering. The second location is within the intralobular bile ducts. The third is peripheral to the bile ducts and is represented by a cell that does not express classical stem cell markers. Cells matching this description have been identified after treatment with agents that induce portal zone necrosis, particularly allyl alcohol.4 The final HSPC compartment identified by Kuwahara et al.'s study lies within the hepatic parenchyma, harboring a cell that resembles a small, transitional hepatocyte. Grisham et al.5 have published several articles on the retrorsine/PHx model, which seems to induce proliferation of this particular cell population. In the current study, Kordes et al. present a series of experiments showing that an isolated population of retinoid storing HSCs are able to give rise to a variety of differentiated progeny within the liver. The experiments are eloquently designed and the data lend themselves to an unambiguous conclusion. Retinoid storing HSCs are able to contribute to liver regeneration and give rise to several lineages of functional cells. Of course, these data are open to the same skepticism that similar cell transplant studies have been subjected to (i.e., potential confounding of data by events such as cell fusion, transdifferentiation, and mesenchymal-to-epithelial transition). It should also be noted that the transplanted cell population has not been meticulously characterized. Given the intimate spatial relationship between previously described hepatic progenitor cell populations and stellate cells, it is critical to exclude the possibility of coselecting these two cell types. It would also be interesting to understand the growth kinetics of the transplanted population, and this effort would be simplified by quantitating the percentage of HSC engraftment. This point is particularly relevant in light of studies published by other groups that indicate that the mode of injury can affect cell transplant efficiency.6 Last, it is important to understand that there are limitations to any cell transplant model. Depending on where the transplanted cells engraft into the recipient liver, alterations in cell-cell and cell-matrix associations can result in a subtly different microenvironment that can affect cell phenotype and lineage potential. It is generally accepted that the body has multiple mechanisms of liver repair. A study published by Wang et al.7 demonstrates that, in a mouse model for tyrosinemia, cell fusion was the predominant mechanism for repair. Data published by Michalopoulos et al.8 indicate that, under certain physiological conditions, mature hepatocytes have the ability to transdifferentiate into cholangiocytes. Additionally, Schaub et al. recently published an article that challenged the long-held tenant that oval/stem cells contribute directly to liver regeneration. In addition, they make the accurate observation that a vast majority of our understanding of hepatic oval/stem cells has been generated in the rat model, but this has not been confirmed in other animal species or humans.9 Nevertheless, it cannot be disputed that a reservoir of cells other than hepatocytes exists that is capable of regenerating liver parenchyma postinjury. It is likely that there are more than one cell type with this capability, and for any specific injury, the easiest repair pathway is utilized. This notion is in good alignment with the principal of Occam's Razor, which states that among a series of potential explanations, the simple answer tends to be the most likely. Several studies on the isolation of liver progenitor cells have targeted different cell populations. The simplest explanation for this fact would be that there are multiple populations of cells that are able to affect repair of the liver. The liver is the only solid organ in the body that has the ability to regenerate itself in response to massive injury. The speed and efficiency of this regeneration is quite remarkable. Throughout the decades since the liver progenitor cell was first described, many reports have been published that identify specific populations with such potential. The article by Kordes et al. clearly describes another multipotent cell population with the potential to give rise to many cell types within the liver. Moving forward, it is becoming increasingly important to define the molecular regulation of these cells and determine their therapeutic potential for cell therapies, tissue engineering, and regenerative medicine. Thomas D. Shupe, Ph.D.1Bryon E. Petersen, Ph.D.2 1Wake Forest Institute for Regenerative Medicine Wake Forest School of Medicine Winston-Salem, NC 2Department of Pediatrics Universty of Florida Gainesville, FL

Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell‐matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter‐driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen‐inducible Cre‐loxP system down‐regulated integrin αvβ6 in DDC‐damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis‐related genes, smaller areas of alpha‐smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)‐β1 activation in vitro . Conclusions : CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF‐β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver disease. (H epatology 2015;61:678‐691)

AIM:To study the binding of connective tissue growth factor (CTGF) to cystine knot-containing ligands and how this impacts platelet-derived growth factor (PDGF)-B signaling.METHODS:The binding strengths of CTGF to cystine knot-containing growth factors including vascular endothelial growth factor (VEGF)-A, PDGF-B, bone morphogenetic protein (BMP)-4, and transforming growth factor (TGF)-β1 were compared using the LexA-based yeast two-hybrid system. EYG48 reporter strain that carried a wild-type LEU2 gene under the control of LexA operators and a lacZ reporter plasmid (p80p-lacZ) containing eight high affinity LexA binding sites were used in the yeast two-hybrid analysis. Interactions between CTGF and the tested growth factors were evaluated based on growth of transformed yeast cells on selective media and colorimetric detection in a liquid β-galactosidase activity assay. Dissociation constants of CTGF to VEGF-A isoform 165 or PDGF-BB homo-dimer were measured in surface plasma resonance (SPR) analysis. CTGF regulation in PDGF-B presentation to the PDGF receptor β (PDGFRβ) was also quantitatively assessed by the SPR analysis. Combinational effects of CTGF protein and PDGF-BB on activation of PDGFRβ and downstream signaling molecules ERK1/2 and AKT were assessed in rabbit corneal fibroblast cells by Western analysis.RESULTS:In the LexA-based yeast two-hybrid system, cystine knot motifs of tested growth factors were fused to the activation domain of the transcriptional factor GAL4 while CTGF was fused to the DNA binding domain of the bacterial repressor protein LexA. Yeast co-transformants containing corresponding fusion proteins for CTGF and all four tested cystine knot motifs survived on selective medium containing galactose and raffinose but lacking histidine, tryptophan, and uracil. In liquid β-galactosidase assays, CTGF expressing cells that were co-transformed with the cystine knot of VEGF-A had the highest activity, at 29.88 ± 0.91 fold above controls (P < 0.01). Cells containing the cystine knot of BMP-4 expressed the second most activity, with a 24.77 ± 0.47 fold increase (P < 0.01). Cells that contained the cystine knot of TGF-β1 had a 3.80 ± 0.66 fold increase (P < 0.05) and the ones with the cystine knot of PDGF-B had a 2.64 ± 0.33 fold increase of β-galactosidase activity (P < 0.01). Further SPR analysis showed that the association rate between VEGF-A 165 and CTGF was faster than PDGF-BB and CTGF. The calculated dissociation constant (KD) of CTGF to VEGF165 and PDGF-BB was 1.8 and 43 nmol/L respectively. PDGF-BB ligand and PDGFRβ receptor formed a stable complex with a low dissociation constant 1.4 nmol/L. Increasing the concentration of CTGF up to 263.2 nmol/L significantly the ligand/receptor binding. In addition, CTGF potentiated phosphorylation of PDGFRβ and AKT in rabbit corneal fibroblast cells stimulated by PDGF-BB in tissue culture condition. In contrast, CTGF did not affect PDGF-B induced phosphorylation of ERK1/2.CONCLUSION:CTGF has a differential binding affinity to VEGF-A, PDGF-B, BMP-4, and TGF-β. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

Hepatic progenitor/oval cell (OC) activation occurs when hepatocyte proliferation is inhibited and is tightly associated with the fibrogenic response during severe Liver damage. Connective tissue growth factor (CTGF) is important for OC activation and contributes to the pathogenesis of Liver fibrosis. By using the Yeast Two-Hybrid approach, we identified a disintegrin and metalloproteinase with thrombospondin repeat 7 (ADAMTS7) as a CTGF binding protein. In vitro characterization demonstrated CTGF binding and processing by ADAMTS7. Moreover, Adamts7 mRNA was induced during OC activation, after the implantation of 2-acetylaminofluorene with partial hepatectomy in rats or on feeding a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet in mice. X-Gal staining showed Adamts7 expression in hepatocyte nuclear factor 4 alpha(+) hepatocytes and desmin(+) myofibroblasts surrounding reactive ducts in DDC-treated Adamts7(-/-) mice carrying a knocked-in LacZ gene. Adamts7 deficiency was associated with higher transcriptional levels of Ctgf and OC markers and enhanced OC proliferation compared to Adamts7(+/+) controls during DDC-induced Liver injury. We also observed increased alpha-smooth muscle actin and procollagen type I mRNAs, large fibrotic areas in alpha-smooth muscle actin and Sirius red staining, and increased production of hepatic collagen by hydroxyproline measurement. These results suggest that ADAMTS7 is a new protease for CTGF protein and a novel regulator in the OC compartment, where its absence causes CTGF accumulation, leading to increased OC activation and biliary fibrosis.

Abstract Introduction: Connective tissue growth factor (CTGF) is a secreted pro-angiogenic protein within the CCN (Cyr61/CTGF/Nov) family and modulates multiple angiogenic pathways through its broad binding ability to cysteine knot motifs presents in key angiogenic factors including vascular endothelial growth factor (VEGF)-A. Overexpression of CTGF has been found in many cancers such as Lewis lung carcinoma (LLC), cholangiocarcinoma (CC), and hepatocellular carcinoma (HCC). In an effort to understand the molecular action of CTGF, we identified a disintegrin and metalloproteinase with thrombospondin type I repeat 7 (ADAMTS7) as a CTGF binding protein in yeast two-hybrid analysis. This enzyme belongs to the ADAMTS family capable of cleaving extracellular matrix (ECM) components and extracellular regulatory molecules. Here, we show that ADAMTS7 binds to and degrades CTGF during cancer development. Methods: Adamts7 knockout mice were used to investigate the importance of ADAMTS7 in CTGF interaction and cleavage during LLC development. Adamts7+/+ and Adamts7−/− mice were subcutaneously implanted with LLC cells. Tumor growth was quantitatively measured within two weeks after implantation. Tumor angiogenesis was assessed utilizing a microvascular density assay. In addition, Adamts7 expression was tracked via X-gal staining of LLC tumors grown in Adamts7−/− mice that contained a LacZ trap-in cassette in the targeted Adamts7 allele. VEGF-A, CTGF, and ADAMTS7 were determined at both the mRNA and protein levels. Results: Adamts7 induction as indicated by the β-galactosidase activity in X-gal staining was found in intra-tumor stromal cells of LLC tumors grown in Adamts7−/− mice. The association of CTGF and ADAMTS7 proteins in protein lysates of LLC tumors grown in Adamts7+/+ mice was confirmed in immunoprecipitation assays. Higher levels of VEGF-A, CTGF and ADAMTS7 mRNAs were found in LLC tumors from Adamts7−/− animals as compared to Adamts7+/+ controls. Slower rate of CTGF turnover, faster growth of LLC tumors, and higher microvascular density were also found in Adamts7−/− animals than controls. Conclusion: These observations confirmed the binding and processing of CTGF by ADAMTS7 during LLC growth. The host-derived ADAMTS7 appears to regulate CTGF turnover and provides a protective effect towards aberrant LLC tumorigenesis and angiogenesis. Citation Format: Liya Pi, Bryon E. Petersen. Interaction between connective tissue growth factor and a disintegrin and metalloproteinase with thrombospondin type I repeats 7 during cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2388. doi:10.1158/1538-7445.AM2015-2388

Tissue engineering and regenerative medicine aim to alleviate the gaps in clinical treatment options for addressing critical defect disorders otherwise left untreated. Ongoing attempts to translate research findings to clinical applications have progressed slower than expected but continue to evolve within the three key areas of cells, signals, and scaffolds. Within these areas, approaches have developed that are evolving our clinical options for addressing disorders as well as improving high-throughout screening techniques to optimize our therapeutic options in tomorrow’s clinic. Such approaches include, but are not limited to, selecting the proper cell sources, engineering artificial and natural biomaterial substrates, bioprinting, microfluidic platforms, and whole organ matrices. Here we review recent advancements in these areas as to better understand where the field is headed.