Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone.

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.

Recombinant CCN1 prevents hyperoxia-induced lung injury in neonatal rats.

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.

Induction of the matricellular protein CCN1 through RhoA and MRTF-A contributes to ischemic cardioprotection.

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.

Degradome products of the matricellular protein CCN1 as modulators of pathological angiogenesis in the retina.

CCN1 is a matricellular protein involved in normal vascular development and tissue repair. CCN1 exhibits cell- and context-dependent activities that are reflective of its tetramodular structure phylogenetically linked to four domains found in various matrix proteins. Here, we show that vitreal fluids from patients with proliferative diabetic retinopathy (PDR) were enriched with a two-module form of CCN1 comprising completely or partially the insulin-like growth factor-binding protein (IGFBP) and von Willebrand factor type C (vWC) domains. The two- and three-module forms comprising, in addition to IGFBP and vWC, the thrombospondin type 1 (TSP1) repeats are CCN1 degradome products by matrix metalloproteinase-2 and -14. The functional significance of CCN1 and its truncated variants was determined in the mouse model of oxygen-induced retinopathy, which simulates neovascular growth associated with PDR and assesses treatment outcomes. In this model, lentivirus-mediated expression of either CCN1 or the IGFBP-vWC-TSP1 form reduced ischemia-induced neovascularization, whereas ectopic expression of the IGFBP-vWC variant exacerbated pathological angiogenesis. The IGFBP-vWC form has potent proangiogenic properties promoting retinal endothelial cell growth, migration, and three-dimensional tubular structure formation, whereas the IGFBP-vWC-TSP1 variant suppressed cell growth and angiogenic gene expression. Both IGFBP-vWC and IGFBP-vWC-TSP1 forms exhibited predictable variations of their domain folding that enhanced their functional potential. These data provide new insights into the formation and activities of CCN1-truncated variants and raise the predictive value of the form containing completely or partially the IGFBP and vWC domains as a surrogate marker of CCN1 activity in PDR distinguishing pathological from physiological angiogenesis.

Role of ß-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury.

Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that ß-catenin signaling promotes epithelial repair after inflammatory injury, but the ß-catenin target genes that mediate this effect are unknown. Herein, we examined which ß-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of ß-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the ß-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that ß-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

FoxM1, a critical regulator of oxidative stress during oncogenesis.

The transcription factor FoxM1 is over-expressed in most human malignancies. Although it is evident that FoxM1 has critical functions in tumour development and progression, the mechanisms by which FoxM1 participates in those processes are not understood. Here, we describe an essential role of FoxM1 in the regulation of oxidative stress that contributes to malignant transformation and tumour cell survival. We identify a negative feedback loop involving FoxM1 that regulates reactive oxygen species (ROS) in proliferating cells. We show that induction of FoxM1 by oncogenic Ras requires ROS. Elevated FoxM1, in turn, downregulates ROS levels by stimulating expression of ROS scavenger genes, such as MnSOD, catalase and PRDX3. FoxM1 depletion sensitizes cells to oxidative stress and increases oncogene-induced premature senescence. Moreover, tumour cells expressing activated AKT1 are 'addicted' to FoxM1, as they require continuous presence of FoxM1 for survival. Together, our results identify FoxM1 as a key regulator of ROS in dividing cells, and provide insights into the mechanism how tumour cells use FoxM1 to control oxidative stress to escape premature senescence and apoptosis.

Analysis of CCN Functions in Liver Regeneration After Partial Hepatectomy.

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.

Atomic Force Microscopy-Based Measurements of Retinal Microvessel Stiffness in Mice with Endothelial-Specific Deletion of CCN1.

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.

Matricellular protein CCN1 activates a proinflammatory genetic program in murine macrophages.

CCN1 (CYR61) is a matricellular protein that is highly expressed at sites of inflammation and wound repair. In these contexts, CCN1 can modify the activities of specific cytokines, enabling TNF-alpha to be cytotoxic without blocking NF-kappaB activity and enhancing the apoptotic activity of Fas ligand and TRAIL. In this paper, we show that CCN1 supports the adhesion of macrophages through integrin alpha(M)beta(2) and syndecan-4, activates NFkappaB-mediated transcription, and induces a proinflammatory genetic program characteristic of classically activated M1 macrophages that participates in Th1 responses. The effects of CCN1 include upregulation of cytokines (TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-12b), chemokines (MIP-1alpha; MCP-3; growth-related oncogenes 1 and 2; and inflammatory protein 10), and regulators of oxidative stress and complement (inducible NO synthase and C3) and downregulation of specific receptors (TLR4 and IL-10Rbeta) and anti-inflammatory factors (TGF-beta1). CCN1 regulates this genetic program through at least two distinct mechanisms: an immediate-early response resulting from direct activation of NF-kappaB by CCN1, leading to the synthesis of cytokines including TNF-alpha and inflammatory protein 10; and a delayed response resulting from CCN1-induced TNF-alpha, which acts as an autocrine/paracrine mediator to activate the expression of other cytokines including IL-1beta and IL-6. These results identify CCN1 as a novel component of the extracellular matrix that activates proinflammatory genes in macrophages, implicating its role in regulating macrophage function during inflammation.

CCN1/CYR61: the very model of a modern matricellular protein.

CCN1 (CYR61) is a dynamically expressed, multifunctional matricellular protein that plays essential roles in cardiovascular development during embryogenesis, and regulates inflammation, wound healing and fibrogenesis in the adult. Aberrant CCN1 expression is associated with myriad pathologies, including various cancers and diseases associated with chronic inflammation. CCN1 promotes diverse and sometimes opposing cellular responses, which can be ascribed, as least in part, to disparate activities mediated through its direct binding to distinct integrins in different cell types and contexts. Accordingly, CCN1 promotes cell proliferation, survival and angiogenesis by binding to integrin α(v)ß(3), and induces apoptosis and senescence through integrin α(6)ß(1) and heparan sulfate proteoglycans. The ability of CCN1 to trigger the accumulation of a robust and sustained level of reactive oxygen species underlies some of its unique activities as a matrix cell-adhesion molecule. Emerging studies suggest that CCN1 might be useful as a biomarker or therapeutic target in certain diseases.

Cellular communication network factor 1-stimulated liver macrophage efferocytosis drives hepatic stellate cell activation and liver fibrosis.

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.

Senescent hepatic stellate cells promote liver regeneration through IL-6 and ligands of CXCR2.

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.

Mechanical regulation of the proangiogenic factor CCN1/CYR61 gene requires the combined activities of MRTF-A and CREB-binding protein histone acetyltransferase.

Smooth muscle-rich tissues respond to mechanical overload by an adaptive hypertrophic growth combined with activation of angiogenesis, which potentiates their mechanical overload-bearing capabilities. Neovascularization is associated with mechanical strain-dependent induction of angiogenic factors such as CCN1, an immediate-early gene-encoded matricellular molecule critical for vascular development and repair. Here we have demonstrated that mechanical strain-dependent induction of the CCN1 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and tethering to a single CArG box sequence within the CCN1 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A(-/-) cells or upon inhibition of CREB-binding protein (CBP) histone acetyltransferase (HAT) either pharmacologically or by siRNAs. Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF-binding site and within the CCN1 gene coding region. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF.MRTF-A complex, whereas enforced induction of p38 by upstream activators (e.g. MKK3 and MKK6) enhanced both CBP HAT and CCN1 promoter activities. Similarly, mechanical overload-induced CCN1 gene expression in vivo was associated with nuclear localization of MRTF-A and enrichment of the CCN1 promoter with both MRTF-A and acetylated histone H3. Taken together, these data suggest that signal-controlled activation of SRF, MRTF-A, and CBP provides a novel connection between mechanical stimuli and angiogenic gene expression.

A conserved phosphorylation site within the forkhead domain of FoxM1B is required for its activation by cyclin-CDK1.

The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G(1)/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G(2)/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G(2)/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1.

The matrix protein CCN1 (CYR61) induces apoptosis in fibroblasts.

Integrin-mediated cell adhesion to extracellular matrix proteins is known to promote cell survival, whereas detachment from the matrix can cause rapid apoptotic death in some cell types. Contrary to this paradigm, we show that fibroblast adhesion to the angiogenic matrix protein CCN1 (CYR61) induces apoptosis, whereas endothelial cell adhesion to CCN1 promotes cell survival. CCN1 induces fibroblast apoptosis through its adhesion receptors, integrin alpha6beta1 and the heparan sulfate proteoglycan (HSPG) syndecan-4, triggering the transcription-independent p53 activation of Bax to render cytochrome c release and activation of caspase-9 and -3. Neither caspase-8 activity nor de novo transcription or translation is required for this process. These results show that cellular interaction with a specific matrix protein can either induce or suppress apoptosis in a cell type-specific manner and that integrin alpha6beta1-HSPGs can function as receptors to induce p53-dependent apoptosis.

Restricting conformational flexibility of the switch II region creates a dominant-inhibitory phenotype in Obg GTPase Nog1.

Nog1 is a conserved eukaryotic GTPase of the Obg family involved in the biogenesis of 60S ribosomal subunits. Here we report the unique dominant-inhibitory properties of a point mutation in the switch II region of mouse Nog1; this mutation is predicted to restrict conformational mobility of the GTP-binding domain. We show that although the mutation does not significantly affect GTP binding, ectopic expression of the mutant in mouse cells disrupts productive assembly of pre-60S subunits and arrests cell proliferation. The mutant impairs processing of multiple pre-rRNA intermediates, resulting in the degradation of the newly synthesized 5.8S/28S rRNA precursors. Sedimentation analysis of nucleolar preribosomes indicates that defective Nog1 function inhibits the conversion of 32S pre-rRNA-containing complexes to a smaller form, resulting in a drastic accumulation of enlarged pre-60S particles in the nucleolus. These results suggest that conformational changes in the switch II element of Nog1 have a critical importance for the dissociation of preribosome-bound factors during intranucleolar maturation and thereby strongly influence the overall efficiency of the assembly process.

CCN1 is an opsonin for bacterial clearance and a direct activator of Toll-like receptor signaling.

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.

Fructose stimulated de novo lipogenesis is promoted by inflammation.

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.

The matricellular protein CCN1 is essential for cardiac development.

The matricellular protein CCN1 (formerly named CYR61) regulates cell adhesion, migration, proliferation, survival, and differentiation through binding to integrin receptors and heparan sulfate proteoglycans. Here we show that Ccn1-null mice are impaired in cardiac valvuloseptal morphogenesis, resulting in severe atrioventricular septal defects (AVSD). Remarkably, haploinsufficiency for Ccn1 also results in delayed formation of the ventricular septum in the embryo and persistent ostium primum atrial septal defects (ASD) in approximately 20% of adults. Mechanistically, Ccn1 is not required for epithelial-to-mesenchymal transformation or cell proliferation and differentiation in the endocardial cushion tissue. However, Ccn1 deficiency leads to precocious apoptosis in the atrial junction of the cushion tissue and impaired gelatinase activities in the muscular component of the interventricular septum at embryonic day 12.5, when fusion between the endocardial cushion tissue and the atrial and ventricular septa occurs, indicating that these defects may underlie the observed AVSD. Moreover, human CCN1 maps to 1p21-p31, the chromosomal location of an AVSD susceptibility gene. Together, these results provide evidence that deficiency in matrix signaling can lead to autosomal dominant AVSD, identify Ccn1(+/-) mice as a genetic model for ostium primum ASD, and implicate CCN1 as a candidate gene for AVSD in humans.