ABSTRACT
The remarkable regenerative capability of the liver has long been appreciated. Upon significant loss of liver tissue, the remnant liver can grow rapidly to restore the original liver mass through a combination of hepatocyte proliferation and hypertrophy to maintain homeostasis. Experimentally, 2/3 partial hepatectomy in mice has been used extensively as a model to dissect the molecular mechanism of liver regeneration and the genetic networks involved. Herein, we describe the protocols for partial hepatectomy and analyses of pertinent CCN protein functions.
Subject(s)
Hepatectomy , Liver Regeneration , Mice , Animals , Hepatectomy/methods , Hepatocytes/metabolism , Liver/surgery , Hyperplasia , Cell ProliferationABSTRACT
Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis.
Subject(s)
Pulse Wave Analysis , Vascular Diseases , Mice , Humans , Animals , Microscopy, Atomic Force , Retina/metabolism , Endothelium , Microvessels , Vascular Diseases/metabolismABSTRACT
Following inflammatory injury in the liver, neutrophils quickly infiltrate the injured tissue to defend against microbes and initiate the repair process; these neutrophils are short lived and rapidly undergo apoptosis. Hepatic stellate cells (HSCs) are the principal precursor cells that transdifferentiate into myofibroblast-like cells, which produce a large amount of extracellular matrix that promotes repair but can also lead to fibrosis if the injury becomes chronic. The matricellular protein cellular communication network factor 1 (CCN1) acts as a bridging molecule by binding phosphatidylserine in apoptotic cells and integrin αv ß3 in phagocytes, thereby triggering efferocytosis or phagocytic clearance of the apoptotic cells. Here, we show that CCN1 induces liver macrophage efferocytosis of apoptotic neutrophils in carbon tetrachloride (CCl4 )-induced liver injury, leading to the production of activated transforming growth factor (TGF)-ß1, which in turn induces HSC transdifferentiation into myofibroblast-like cells that promote fibrosis development. Consequently, knock-in mice expressing a single amino acid substitution in CCN1 rendering it unable to bind αv ß3 or induce efferocytosis are impaired in neutrophil clearance, production of activated TGF-ß1, and HSC transdifferentiation, resulting in greatly diminished liver fibrosis following exposure to CCl4 . Conclusion: These results reveal the crucial role of CCN1 in stimulating liver macrophage clearance of apoptotic neutrophils, a process that drives HSC transdifferentiation into myofibroblastic cells and underlies fibrogenesis in chronic liver injury.
Subject(s)
Hepatic Stellate Cells , Transforming Growth Factor beta1 , Animals , Carbon Tetrachloride/toxicity , Hepatic Stellate Cells/metabolism , Integrins/metabolism , Kupffer Cells/metabolism , Liver Cirrhosis/chemically induced , Mice , Phosphatidylserines , Transforming Growth FactorsABSTRACT
Senescent cells have long been associated with deleterious effects in aging-related pathologies, although recent studies have uncovered their beneficial roles in certain contexts, such as wound healing. We have found that hepatic stellate cells (HSCs) underwent senescence within 2 days after 2/3 partial hepatectomy (PHx) in young (2-3 months old) mice, and the elimination of these senescent cells by using the senolytic drug ABT263 or by using a genetic mouse model impaired liver regeneration. Senescent HSCs secrete IL-6 and CXCR2 ligands as part of the senescence-associated secretory phenotype, which induces multiple signaling pathways to stimulate liver regeneration. IL-6 activates STAT3, induces Yes-associated protein (YAP) activation through SRC family kinases, and synergizes with CXCL2 to activate ERK1/2 to stimulate hepatocyte proliferation. The administration of either IL-6 or CXCL2 partially restored liver regeneration in mice with senescent cell elimination, and the combination of both fully restored liver weight recovery. Furthermore, the matricellular protein central communication network factor 1 (CCN1, previously called CYR61) was rapidly elevated in response to PHx and induced HSC senescence. Knockin mice expressing a mutant CCN1 unable to bind integrin α6ß1 were deficient in senescent cells and liver regeneration after PHx. Thus, HSC senescence, largely induced by CCN1, is a programmed response to PHx and plays a critical role in liver regeneration through signaling pathways activated by IL-6 and ligands of CXCR2.
Subject(s)
Hepatic Stellate Cells , Liver Regeneration , Animals , Hepatectomy , Hepatic Stellate Cells/metabolism , Interleukin-6/metabolism , Ligands , Liver Regeneration/physiology , Mice , Receptors, Interleukin-8BABSTRACT
Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.
Subject(s)
Fructose/pharmacology , Inflammation/metabolism , Lipogenesis/drug effects , Acetyl Coenzyme A/pharmacology , Animals , Endotoxemia/blood , Female , Fructosephosphates/pharmacology , Gastrointestinal Microbiome , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Intestines/drug effects , Lipidomics , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/metabolism , Regeneration/drug effects , Toll-Like Receptors/agonistsABSTRACT
Expression of the matricellular protein CCN1 (CYR61) is associated with inflammation and is required for successful wound repair. Here, we show that CCN1 binds bacterial pathogen-associated molecular patterns including peptidoglycans of Gram-positive bacteria and lipopolysaccharides of Gram-negative bacteria. CCN1 opsonizes methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa and accelerates their removal by phagocytosis and increased production of bactericidal reactive oxygen species in macrophages through the engagement of integrin αvß3. Mice with myeloid-specific Ccn1 deletion and knock-in mice expressing CCN1 unable to bind αvß3 are more susceptible to infection by S. aureus or P. aeruginosa, resulting in increased mortality and organ colonization. Furthermore, CCN1 binds directly to TLR2 and TLR4 to activate MyD88-dependent signaling, cytokine expression and neutrophil mobilization. CCN1 is therefore a pattern recognition receptor that opsonizes bacteria for clearance and functions as a damage-associated molecular pattern to activate inflammatory responses, activities that contribute to wound healing and tissue repair.
Subject(s)
Cysteine-Rich Protein 61/metabolism , Opsonin Proteins/metabolism , Pseudomonas Infections/immunology , Staphylococcal Infections/immunology , Toll-Like Receptors/metabolism , Animals , Cysteine-Rich Protein 61/genetics , Cysteine-Rich Protein 61/immunology , Disease Models, Animal , Disease Susceptibility , Female , Gene Knock-In Techniques , Gene Knockdown Techniques , Humans , Integrin alphaVbeta3/immunology , Integrin alphaVbeta3/metabolism , Male , Methicillin-Resistant Staphylococcus aureus , Mice , Mice, Inbred C57BL , Mice, Transgenic , Opsonin Proteins/genetics , Pathogen-Associated Molecular Pattern Molecules/metabolism , Phagocytosis/immunology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/immunology , Sf9 Cells , Signal Transduction/immunology , Staphylococcal Infections/microbiology , Toll-Like Receptors/immunologyABSTRACT
Adult neural stem cells (NSCs) reside in specialized niches, which hold a balanced number of NSCs, their progeny, and other cells. How niche capacity is regulated to contain a specific number of NSCs remains unclear. Here, we show that ependyma-derived matricellular protein CCN1 (cellular communication network factor 1) negatively regulates niche capacity and NSC number in the adult ventricular-subventricular zone (V-SVZ). Adult ependyma-specific deletion of Ccn1 transiently enhanced NSC proliferation and reduced neuronal differentiation in mice, increasing the numbers of NSCs and NSC units. Although proliferation of NSCs and neurogenesis seen in Ccn1 knockout mice eventually returned to normal, the expanded NSC pool was maintained in the V-SVZ until old age. Inhibition of EGFR signaling prevented expansion of the NSC population observed in CCN1 deficient mice. Thus, ependyma-derived CCN1 restricts NSC expansion in the adult brain to maintain the proper niche capacity of the V-SVZ.
Subject(s)
Cysteine-Rich Protein 61/metabolism , Neurogenesis/physiology , Signal Transduction , Adult Stem Cells/physiology , Animals , Brain , Cysteine-Rich Protein 61/genetics , Ependyma/cytology , Ependyma/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Stem Cells/cytology , Neural Stem Cells/metabolismSubject(s)
Cell Proliferation , Cellular Senescence , Cysteine-Rich Protein 61/metabolism , Fibroblasts/metabolism , Heart Injuries/metabolism , Myocytes, Cardiac/metabolism , Regeneration , Aniline Compounds/pharmacology , Animals , Cell Adhesion Molecules/deficiency , Cell Adhesion Molecules/genetics , Cell Proliferation/drug effects , Cellular Senescence/drug effects , Cysteine-Rich Protein 61/genetics , Disease Models, Animal , Fibroblasts/drug effects , Fibroblasts/pathology , Fibrosis , Heart Injuries/genetics , Heart Injuries/pathology , Heart Injuries/physiopathology , Mice, Knockout , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Paracrine Communication , Regeneration/drug effects , Signal Transduction , Sulfonamides/pharmacology , Time Factors , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/geneticsABSTRACT
Fibrosis is the excessive accumulation of extracellular matrix that often occurs as a wound healing response to repeated or chronic tissue injury, and may lead to the disruption of organ architecture and loss of function. Although fibrosis was previously thought to be irreversible, recent evidence indicates that certain circumstances permit the resolution of fibrosis when the underlying causes of injury are eradicated. The mechanism of fibrosis resolution encompasses degradation of the fibrotic extracellular matrix as well as elimination of fibrogenic myofibroblasts through their adaptation of various cell fates, including apoptosis, senescence, dedifferentiation, and reprogramming. In this Review, we discuss the present knowledge and gaps in our understanding of how matrix degradation is regulated and how myofibroblast cell fates can be manipulated, areas that may identify potential therapeutic approaches for fibrosis.
Subject(s)
Apoptosis , Cell Dedifferentiation , Cellular Reprogramming , Cellular Senescence , Extracellular Matrix/metabolism , Myofibroblasts/metabolism , Animals , Extracellular Matrix/pathology , Fibrosis , Humans , Myofibroblasts/pathologyABSTRACT
The expression of Ccn1 (Cyr61) is essential for cardiovascular development during embryogenesis, whereas in adulthood it is associated with inflammation, wound healing, injury repair, and related pathologies including fibrosis and cancer. Recent studies have found that CCN1 plays a critical role in promoting wound healing and tissue repair. Mechanistically, CCN1 functions through direct interaction with specific integrin receptors expressed in various cell types in the wound tissue microenvironment to coordinate diverse cellular functions for repair. Here we briefly summarize the current knowledge on the functions of CCN1 in tissue injury repair and discuss pertinent unanswered questions.
ABSTRACT
BackgroundCystein-rich protein 61 (Cyr61/CCN1) is a member of the CCN family of matricellular proteins that has an important role in tissue development and remodeling. However, the role of CCN1 in the pathogenesis of bronchopulmonary dysplasia (BPD) is unknown. Accordingly, we have investigated the effects of CCN1 on a hyperoxia-induced lung injury model in neonatal rats.MethodsIn experiment 1, newborn rats were randomized to room air (RA) or 85% oxygen (O2) for 7 or 14 days, and we assessed the expression of CCN1. In experiment 2, rat pups were exposed to RA or O2 and received placebo or recombinant CCN1 by daily intraperitoneal injection for 10 days. The effects of CCN1 on hyperoxia-induced lung inflammation, alveolar and vascular development, vascular remodeling, and right ventricular hypertrophy (RVH) were observed.ResultsIn experiment 1, hyperoxia downregulated CCN1 expression. In experiment 2, treatment with recombinant CCN1 significantly decreased macrophage and neutrophil infiltration, reduced inflammasome activation, increased alveolar and vascular development, and reduced vascular remodeling and RVH in the hyperoxic animals.ConclusionThese results demonstrate that hyperoxia-induced lung injury is associated with downregulated basal CCN1 expression, and treatment with CCN1 can largely reverse hyperoxic injury.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Bronchopulmonary Dysplasia/prevention & control , Cysteine-Rich Protein 61/pharmacology , Hyperoxia/complications , Lung Injury/prevention & control , Lung/drug effects , Pulmonary Artery/drug effects , Animals , Animals, Newborn , Bronchopulmonary Dysplasia/etiology , Bronchopulmonary Dysplasia/metabolism , Bronchopulmonary Dysplasia/pathology , Cysteine-Rich Protein 61/genetics , Cysteine-Rich Protein 61/metabolism , Disease Models, Animal , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/prevention & control , Inflammasomes/drug effects , Inflammasomes/metabolism , Lung/metabolism , Lung/pathology , Lung Injury/etiology , Lung Injury/metabolism , Lung Injury/pathology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Neovascularization, Physiologic/drug effects , Neutrophil Infiltration/drug effects , Pneumonia/etiology , Pneumonia/prevention & control , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Time Factors , Vascular Remodeling/drug effectsABSTRACT
CYR61-CTGF-NOV (CCN)1 is a dynamically expressed extracellular matrix (ECM) protein with critical functions in cardiovascular development and tissue repair. Angiogenic endothelial cells (ECs) are a major cellular source of CCN1 which, once secreted, associates with the ECM and the cell surface and tightly controls the bidirectional flow of information between cells and the surrounding matrix. Endothelium-specific CCN1 deletion in mice using a cre/lox strategy induces EC hyperplasia and causes blood vessels to coalesce into large flat hyperplastic sinuses with no distinctive hierarchical organization. This is consistent with the role of CCN1 as a negative feedback regulator of vascular endothelial growth factor (VEGF) receptor activation. In the mouse model of oxygen-induced retinopathy (OIR), pericytes become the predominant CCN1 producing cells. Pericyte-specific deletion of CCN1 significantly decreases pathological retinal neovascularization following OIR. CCN1 induces the expression of the non-canonical Wnt5a in pericyte but not in EC cultures. In turn, exogenous Wnt5a inhibits CCN1 gene expression, induces EC proliferation and increases hypersprouting. Concordantly, treatment of mice with TNP470, a non-canonical Wnt5a inhibitor, reestablishes endothelial expression of CCN1 and significantly decreases pathological neovascular growth in OIR. Our data highlight the significance of CCN1-EC and CCN1-pericyte communication signals in driving physiological and pathological angiogenesis.
Subject(s)
Cysteine-Rich Protein 61/metabolism , Endothelial Cells/metabolism , Pericytes/metabolism , Retinal Neovascularization/metabolism , Wnt-5a Protein/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Ischemia/complications , Mice, Inbred C57BL , Retinal Neovascularization/etiology , Wnt Signaling PathwayABSTRACT
The embryonic lethality of mice with conventional global knockout of Ccn1 (Cyr61) precludes analysis of Ccn1 functions in late embryonic development or in adulthood. To circumvent this limitation, we have generated conditional knockout mice that allow cell type-specific deletion of Ccn1, and constructed an allelic series of Ccn1 knockin mice that express CCN1 defective for binding specific integrins in lieu of the wild type protein. Here we describe the construction of these mice and discuss how analysis of these animals can provide unique insights into Ccn1 functions mediated through specific integrin receptors. It is anticipated that future analysis of mice carrying specific mutations in genes of the Ccn family will be greatly facilitated by application of the CRISPR/Cas9 gene editing methodology.
Subject(s)
Alleles , Cysteine-Rich Protein 61/genetics , Animals , Cysteine-Rich Protein 61/metabolism , Embryonic Stem Cells , Fibroblasts , Gene Order , Gene Targeting , Genetic Loci , Mice , Mice, Transgenic , Mutation , Plasmids/geneticsABSTRACT
The expression of Ccn2 (CTGF) has been linked to fibrosis in many tissues and pathologies, although its activities in fibroblastic cells and precise mechanism of action in fibrogenesis are still controversial. Here, we showed that CCN2 can induce cellular senescence in fibroblasts both in vitro and in vivo, whereupon senescent cells express an anti-fibrotic "senescence-associated secretory phenotype" (SASP) that includes upregulation of matrix metalloproteinases and downregulation of collagen. Mechanistically, CCN2 induces fibroblast senescence through integrin α6ß1-mediated accumulation of reactive oxygen species, leading to activation of p53 and induction of p16INK4a. In cutaneous wound healing, Ccn2 expression is highly elevated only during the initial inflammatory phase and quickly declines thereafter to a low level during the proliferation and maturation phases of healing when myofibroblasts play a major role. Consistent with this expression kinetics, knockdown of Ccn2 has little effect on the rate of wound closure, formation of senescent cells, or collagen content of the wounds. However, application of purified CCN2 protein on cutaneous wounds leads to induction of senescent cells, expression of SASP, and reduction of collagen content. These results show that CCN2 can induce cellular senescence in fibroblasts and is capable of exerting an anti-fibrotic effect in a context-dependent manner.
ABSTRACT
The CCN family (CYR61; CTGF; NOV; CCN1-6; WISP1-3) of matricellular proteins in mammals is comprised of six homologous members that play important roles in development, inflammation, tissue repair, and a broad range of pathological processes including fibrosis and cancer. Despite considerable effort to search for a high affinity CCN-specific receptor akin to growth factor receptors, no such receptor has been found. Rather, CCNs bind several groups of multi-ligand receptors as characteristic of other matricellular proteins. The most extensively documented among CCN-binding receptors are integrins, including αvß3, αvß5, α5ß1, α6ß1, αIIbß3, αMß2, and αDß2, which mediate diverse CCN functions in various cell types. CCNs also bind cell surface heparan sulfate proteoglycans (HSPGs), low density liproprotein receptor-related proteins (LRPs), and the cation-independent mannose-6-phosphate (M6P) receptor, which are endocytic receptors that may also serve as co-receptors in cooperation with other cell surface receptors. CCNs have also been reported to bind FGFR-2, Notch, RANK, and TrkA, potentially altering the affinities of these receptors for their ligands. The ability of CCNs to bind a multitude of receptors in various cell types may account for the remarkable versatility of their functions, and underscore the diverse signaling pathways that mediate their activities.
Subject(s)
Biliary Tract/physiology , Cysteine-Rich Protein 61/metabolism , Liver/physiology , Regeneration/physiology , Wound Healing/physiology , Animals , Biliary Tract/injuries , Carcinoma, Hepatocellular/pathology , Humans , Liver/injuries , Liver Cirrhosis/pathology , Liver Neoplasms/pathology , MiceABSTRACT
Neutrophil infiltration constitutes the first step in wound healing, although their timely clearance by macrophage engulfment, or efferocytosis, is critical for efficient tissue repair. However, the specific mechanism for neutrophil clearance in wound healing remains undefined. Here we uncover a key role for CCN1 in neutrophil efferocytosis by acting as a bridging molecule that binds phosphatidylserine, the 'eat-me' signal on apoptotic cells and integrins αvß3/αvß5 in macrophages to trigger efferocytosis. Both knockin mice expressing a mutant CCN1 that is unable to bind αvß3/αvß5 and mice with Ccn1 knockdown are defective in neutrophil efferocytosis, resulting in exuberant neutrophil accumulation and delayed healing. Treatment of wounds with CCN1 accelerates neutrophil clearance in both Ccn1 knockin mice and diabetic Lepr(db/db) mice, which suffer from neutrophil persistence and impaired healing. These findings establish CCN1 as a critical opsonin in skin injury and suggest a therapeutic potential for CCN1 in certain types of non-healing wounds.
Subject(s)
Cysteine-Rich Protein 61/genetics , Cytophagocytosis/genetics , Macrophages/immunology , Neutrophils/immunology , Skin/injuries , Wound Healing/genetics , Animals , Cell Migration Assays , Cysteine-Rich Protein 61/immunology , Cysteine-Rich Protein 61/pharmacology , Cytophagocytosis/drug effects , Cytophagocytosis/immunology , Diabetes Mellitus/genetics , Diabetes Mellitus/immunology , Epidermal Growth Factor/pharmacology , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Keratinocytes/drug effects , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Neutrophils/drug effects , Real-Time Polymerase Chain Reaction , Receptors, Leptin/genetics , Receptors, Vitronectin , Reverse Transcriptase Polymerase Chain Reaction , Skin/drug effects , Skin/immunology , Wound Healing/drug effects , Wound Healing/immunologyABSTRACT
Liver cholestatic diseases, which stem from diverse etiologies, result in liver toxicity and fibrosis and may progress to cirrhosis and liver failure. We show that CCN1 (also known as CYR61), a matricellular protein that dampens and resolves liver fibrosis, also mediates cholangiocyte proliferation and ductular reaction, which are repair responses to cholestatic injury. In cholangiocytes, CCN1 activated NF-κB through integrin αvß5/αvß3, leading to Jag1 expression, JAG1/NOTCH signaling, and cholangiocyte proliferation. CCN1 also induced Jag1 expression in hepatic stellate cells, whereupon they interacted with hepatic progenitor cells to promote their differentiation into cholangiocytes. Administration of CCN1 protein or soluble JAG1 induced cholangiocyte proliferation in mice, which was blocked by inhibitors of NF-κB or NOTCH signaling. Knock-in mice expressing a CCN1 mutant that is unable to bind αvß5/αvß3 were impaired in ductular reaction, leading to massive hepatic necrosis and mortality after bile duct ligation (BDL), whereas treatment of these mice with soluble JAG1 rescued ductular reaction and reduced hepatic necrosis and mortality. Blockade of integrin αvß5/αvß3, NF-κB, or NOTCH signaling in WT mice also resulted in defective ductular reaction after BDL. These findings demonstrate that CCN1 induces cholangiocyte proliferation and ductular reaction and identify CCN1/αvß5/NF-κB/JAG1 as a critical axis for biliary injury repair.
Subject(s)
Bile Ducts/metabolism , Cysteine-Rich Protein 61/physiology , Liver/metabolism , NF-kappa B/metabolism , Receptors, Vitronectin/physiology , Animals , Bile Ducts/physiology , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/pharmacology , Calcium-Binding Proteins/therapeutic use , Cell Division , Cells, Cultured , Cholestasis, Extrahepatic/genetics , Cholestasis, Extrahepatic/metabolism , Cholestasis, Extrahepatic/pathology , Cysteine-Rich Protein 61/genetics , Cysteine-Rich Protein 61/pharmacology , Gene Expression Regulation , Gene Knock-In Techniques , Hepatic Stellate Cells/metabolism , Hepatocytes/metabolism , Humans , Integrin alphaVbeta3 , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/pharmacology , Intercellular Signaling Peptides and Proteins/therapeutic use , Jagged-1 Protein , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Membrane Proteins/therapeutic use , Mice , Mice, Inbred C57BL , RNA Interference , Receptors, Notch/physiology , Recombinant Fusion Proteins/metabolism , Regeneration , Serrate-Jagged ProteinsABSTRACT
Endometriosis is a prevalent gynecological disorder in which endometrial tissue proliferates in extrauterine sites, such as the peritoneal cavity, eventually giving rise to painful, invasive lesions. Dysregulated estradiol (E) signaling has been implicated in this condition. However, the molecular mechanisms that operate downstream of E in the ectopic endometrial tissue are unknown. To investigate these mechanisms, we used a mouse model of endometriosis. Endometrial tissue from donor mice was surgically transplanted on the peritoneal surface of immunocompetent syngeneic recipient mice, leading to the establishment of cystic endometriosis-like lesions. Our studies revealed that treatment with E led to an approximately 3-fold increase in the lesion size within a week of transplantation. E also caused a concomitant stimulation in the expression of connective tissue growth factor/Cyr61/Nov (CCN1), a secreted cysteine-rich matricellular protein, in the lesions. Interestingly, CCN1 is highly expressed in human ectopic endometriotic lesions. To address its role in endometriosis, endometrial tissue from Ccn1-null donor mice was transplanted in wild-type recipient mice. The resulting ectopic lesions were reduced up to 75% in size compared with wild-type lesions due to diminished cell proliferation and cyst formation. Notably, loss of CCN1 also disrupted the development of vascular networks in the ectopic lesions and reduced the expression of several angiogenic factors, such as vascular endothelial growth factor-A and vascular endothelial growth factor-C. These results suggest that CCN1, acting downstream of E, critically controls cell proliferation and neovascularization, which support the growth and survival of endometriotic tissue at ectopic sites. Blockade of CCN1 signaling during the early stages of lesion establishment may provide a therapeutic avenue to control endometriosis.
Subject(s)
Cysteine-Rich Protein 61/metabolism , Endometriosis/metabolism , Endometriosis/pathology , Estrogens/pharmacology , Animals , Cysteine-Rich Protein 61/genetics , Disease Models, Animal , Female , Immunohistochemistry , Mice , Real-Time Polymerase Chain ReactionABSTRACT
Activation of RhoA, a low molecular-weight G-protein, plays an important role in protecting the heart against ischemic stress. Studies using non-cardiac cells demonstrate that the expression and subsequent secretion of the matricellular protein CCN1 is induced by GPCR agonists that activate RhoA. In this study we determined whether and how CCN1 is induced by GPCR agonists in cardiomyocytes and examined the role of CCN1 in ischemic cardioprotection in cardiomyocytes and the isolated perfused heart. In neonatal rat ventricular myocytes (NRVMs), sphingosine 1-phosphate (S1P), lysophosphatidic acid (LPA) and endothelin-1 induced robust increases in CCN1 expression while phenylephrine, isoproterenol and carbachol had little or no effect. The ability of agonists to activate the small G-protein RhoA correlated with their ability to induce CCN1. CCN1 induction by S1P was blocked when RhoA function was inhibited with C3 exoenzyme or a pharmacological RhoA inhibitor. Conversely overexpression of RhoA was sufficient to induce CCN1 expression. To delineate the signals downstream of RhoA we tested the role of MRTF-A (MKL1), a co-activator of SRF, in S1P-mediated CCN1 expression. S1P increased the nuclear accumulation of MRTF-A and this was inhibited by the functional inactivation of RhoA. In addition, pharmacological inhibitors of MRTF-A or knockdown of MRTF-A significantly diminished S1P-mediated CCN1 expression, indicating a requirement for RhoA/MRTF-A signaling. We also present data indicating that CCN1 is secreted following agonist treatment and RhoA activation, and binds to cells where it can serve an autocrine function. To determine the functional significance of CCN1 expression and signaling, simulated ischemia/reperfusion (sI/R)-induced apoptosis was assessed in NRVMs. The ability of S1P to protect against sI/R was significantly reduced by the inhibition of RhoA, ROCK or MRTF-A or by CCN1 knockdown. We also demonstrate that ischemia/reperfusion induces CCN1 expression in the isolated perfused heart and that this functions as a cardioprotective mechanism, evidenced by the significant increase in infarct development in response to I/R in the cardiac specific CCN1 KO relative to control mice. Our findings implicate CCN1 as a mediator of cardioprotection induced by GPCR agonists that activate RhoA/MRTF-A signaling.