G-quadruplexes Stabilization Upregulates CCN1 and Accelerates Aging in Cultured Cerebral Endothelial Cells

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. However, whether CCN1 contributes to senescence in CEC and how this is regulated requires further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. Stabilization of the G4 structures regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and these mice exhibited enhanced cognitive deficits. A sequence in the 3′-UTR of the human CCN1 mRNA has the ability to fold into G4s in vitro. We hypothesize that G4 stabilization regulates CCN1 in cultured primary CEC and induces endothelial senescence. We used cerebral microvessel fractions and cultured primary CEC from young (4-months old, m/o) and aged (18-m/o) mice to determine CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA-G4s for fluorescence spectra. We found that cultured CEC from aged mice exhibited enhanced levels of SA phenotypes, and higher levels of CCN1 and G4 stabilization. In cultured CEC, CCN1 induced SA phenotypes, such as SA β-galactosidase activity, and double-strand DNA damage. Furthermore, CCN1 levels were upregulated by a G4 ligand, and a G-rich motif in the 3′-UTR of the Ccn1 mRNA was folded into a G4. In conclusion, we demonstrate that CCN1 can induce senescence in cultured primary CEC, and we provide evidence that G4 stabilization is a novel mechanism regulating the SASP component CCN1.

CCN2 (Cellular Communication Network Factor 2) Deletion Alters Vascular Integrity and Function Predisposing to Aneurysm Formation

Background: CCN2 (cellular communication network factor 2) is a matricellular protein involved in cell communication and microenvironmental signaling responses. CCN2 is known to be overexpressed in several cardiovascular diseases, but its role is not completely understood. Methods: Here, CCN2 involvement in aortic wall homeostasis and response to vascular injury was investigated in inducible Ccn2 -deficient mice, with induction of vascular damage by infusion of Ang II (angiotensin II; 15 days), which is known to upregulate CCN2 expression in the aorta. Results: Ang II infusion in CCN2-silenced mice lead to 60% mortality within 10 days due to rapid development and rupture of aortic aneurysms, as evidenced by magnetic resonance imaging, echography, and histological examination. Ccn2 deletion decreased systolic blood pressure and caused aortic structural and functional changes, including elastin layer disruption, smooth muscle cell alterations, augmented distensibility, and increased metalloproteinase activity, which were aggravated by Ang II administration. Gene ontology analysis of RNA sequencing data identified aldosterone biosynthesis as one of the most enriched terms in CCN2-deficient aortas. Consistently, treatment with the mineralocorticoid receptor antagonist spironolactone before and during Ang II infusion reduced aneurysm formation and mortality, underscoring the importance of the aldosterone pathway in Ang II–induced aorta pathology. Conclusions: CCN2 is critically involved in the functional and structural homeostasis of the aorta and in maintenance of its integrity under Ang II–induced stress, at least, in part, by disruption of the aldosterone pathway. Thus, this study opens new avenues to future studies in disorders associated to vascular pathologies.

CCN1 coordinately regulates intestinal stem cell proliferation and differentiation through integrins αvβ3/αvβ5

Intestinal stem cells (ISCs) at the crypt base contribute to intestinal homeostasis through a balance between self-renewal and differentiation. However, the molecular mechanisms regulating this homeostatic balance remain elusive. Here we show that the matricellular protein CCN1/CYR61 coordinately regulates ISC proliferation and differentiation through distinct pathways emanating from CCN1 interaction with integrins αvβ3/αvβ5. Mice that delete Ccn1 in Lgr5+ ISCs or express mutant CCN1 unable to bind integrins αvβ3/αvβ5 exhibited exuberant ISC expansion and enhanced differentiation into secretory cells at the expense of absorptive enterocytes in the small intestine, leading to nutrient malabsorption. Analysis of crypt organoids revealed that through integrins αvβ3/αvβ5, CCN1 induces NF-κB-dependent Jag1 expression to regulate Notch activation for differentiation and promotes Src-mediated YAP activation and Dkk1 expression to control Wnt signaling for proliferation. Moreover, CCN1 and YAP amplify the activities of each other in a regulatory loop. These findings establish CCN1 as a novel niche factor in the intestinal crypts, providing new insights into how matrix signaling exerts overarching control of ISC homeostasis.

Metabolic Effects of CCN5/WISP2 Gene Deficiency and Transgenic Overexpression in Mice

CCN5/WISP2 is a matricellular protein, the expression of which is under the regulation of Wnt signaling and IGF-1. Our initial characterization supports the notion that CCN5 might promote the proliferation and survival of pancreatic β-cells and thus improve the metabolic profile of the animals. More recently, the roles of endogenous expression of CCN5 and its ectopic, transgenic overexpression on metabolic regulation have been revealed through two reports. Here, we attempt to compare the experimental findings from those studies, side-by-side, in order to further establish its roles in metabolic regulation. Prominent among the discoveries was that a systemic deficiency of CCN5 gene expression caused adipocyte hypertrophy, increased adipogenesis, and lipid accumulation, resulting in insulin resistance and glucose intolerance, which were further exacerbated upon high-fat diet feeding. On the other hand, the adipocyte-specific and systemic overexpression of CCN5 caused an increase in lean body mass, improved insulin sensitivity, hyperplasia of cardiomyocytes, and increased heart mass, but decreased fasting glucose levels. CCN5 is clearly a regulator of adipocyte proliferation and maturation, affecting lean/fat mass ratio and insulin sensitivity. Not all results from these models are consistent; moreover, several important aspects of CCN5 physiology are yet to be explored.

Matricellular Protein Periostin Promotes Pericyte Migration in Fibrotic Airways

Introduction: Periostin is a matricellular protein that is currently used as a biomarker for asthma. However, its contribution to tissue remodeling in allergic asthma is currently unknown. We have previously demonstrated that tissue-resident mesenchymal stem cells known as pericytes are a key cell type involved in airway remodeling. This is thought to be caused the uncoupling of pericytes from the microvasculature supporting the large airways, facilitated by inflammatory growth factors and cytokines. It is hypothesized that periostin may be produced by profibrotic pericytes and contribute to the remodeling observed in allergic asthma.Methods: Lung sections from mice with allergic airway disease driven by exposure to house dust mite (HDM) were stained using an anti-periostin antibody to explore its involvement in fibrotic lung disease. Human pericytes were cultured in vitro and stained for periostin to assess periostin expression. Migration assays were performed using human pericytes that were pretreated with TGF-β or periostin. ELISAs were also carried out to assess periostin expression levels in bronchoalveolar lavage fluid as well as the induction of periostin production by IL-13.Results: Immunostaining indicated that pericytes robustly express periostin, with increased expression following treatment with TGF-β. Migration assays demonstrated that pericytes treated with periostin were more migratory. Periostin production was also increased in HDM exposed mice as well as in cultured pericytes treated with IL-13.Conclusion: Periostin is produced by pericytes in response to TGF-β or IL-13, and periostin plays a key role in inducing pericyte migration. The increase in periostin expression in TGF-β or IL-13 treated pericytes suggests that IL-13 may trigger periostin production in pericytes whilst TGF-β modulates periostin expression to promote pericyte migration in the context of tissue fibrosis.

Following the SPARC: tracking Secreted Protein Acidic and Rich in Cysteine associated with LX-2´s extracellular vesicles as a non-destructive modality to evaluate lipid-based antifibrotic treatments

Uncovering the complex cellular mechanisms underlying hepatic fibrogenesis, a highly dynamic and active process ultimately responsible for liver failure if left untreated, could expedite the development of effective treatments and noninvasive diagnostic modalities for this often silent pathology. The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers that might become a viable alternative to invasive liver biopsies. We developed a solid set of methods to isolate and characterize EVs from differently treated human hepatic stellate cell (HSC) line LX-2 in vitro, and we investigated the biological effect they exert onto naïve LX-2, proving that EVs do play an active role in fibrogenesis. Electrical/asymmetric flow field-flow fractionation (EAF4) revealed EV subpopulations with different physicochemical behaviors. Proteomic data from our samples was mined for EV-associated proteins whose expression correlated with HSC treatment. Consequently, we chose the secreted protein acidic and cysteine rich (SPARC), a matricellular protein previously reported to be upregulated in activated HSCs, as a proof-of-concept protein to explore the feasibility of using fluorescence nanoparticle tracking analysis as a non-destructive tool for the determination of HSCs’ fibrogenic phenotype based on EVs. We could thus use EVs to directly evaluate the efficacy of treatment with S80, a lipid rich (>75 %) in polyenylphosphatidylcholines (PPC). We found that PPC-rich S80 reduces the relative presence of SPARC-positive EVs. For the first time, we could correlate the cellular response to lipid-based antifibrotic treatment to the relative presence of a candidate protein marker associated with the released EVs. In addition to providing novel insights into PPC treatments, our findings pave the way for more precise and less invasive diagnostic analyses of hepatic fibrogenesis.

Potential Role of CCN Proteins in Breast Cancer: Therapeutic Advances and Perspectives

CCNs are a specific type of matricellular protein, which are essential signaling molecules, and play multiple roles in multicellular eukaryotes. This family of proteins consists of six separate members, which exist only in vertebrates. The architecture of CCN proteins is multi-modular comprising four distinct modules. CCN Proteins achieve their primary functional activities by binding with several integrin7 receptors. The CCN family has been linked to cell adhesion, chemotaxis and migration, mitogenesis, cell survival, angiogenesis, differentiation, tumorigenesis, chondrogenesis, and wound healing, among other biological interactions. Breast cancer is the most commonly diagnosed cancer worldwide and CCN regulated breast cancer stands at the top. A favorable or unfavorable association between various CCNs has been reported in patients with breast carcinomas. The pro-tumorigenic CCN1, CCN2, CCN3, and CCN4 may lead to human breast cancer, although the anti-tumorigenic actions of CCN5 and CCN6 are also present. Several studies have been conducted on CCN proteins and cancer in recent years. CCN1 and CCN3 have been shown to exhibit a dual nature of tumor inhibition and tumor suppression to some extent in quiet recent time. Pharmacological advances in treating breast cancer by targeting CCN proteins are also reported. In our study, we intend to provide an overview of these research works while keeping breast cancer in focus. This information may facilitate early diagnosis, early prognosis and the development of new therapeutic strategies.

Periostin gene expression in neu-positive breast cancer cells is regulated by a FGFR signaling cross talk with TGFβ/PI3K/AKT pathways

Background Breast cancer is a highly heterogeneous disease with multiple drivers and complex regulatory networks. Periostin (Postn) is a matricellular protein involved in a plethora of cancer types and other diseases. Postn has been shown to be involved in various processes of tumor development, such as angiogenesis, invasion, cell survival and metastasis. The expression of Postn in breast cancer cells has been correlated with a more aggressive phenotype. Despite extensive research, it remains unclear how epithelial cancer cells regulate Postn expression. Methods Using murine tumor models and human TMAs, we have assessed the proportion of tumor samples that have acquired Postn expression in tumor cells. Using biochemical approaches and tumor cell lines derived from Neu+ murine primary tumors, we have identified major regulators of Postn gene expression in breast cancer cell lines. Results Here, we show that, while the stromal compartment typically always expresses Postn, about 50% of breast tumors acquire Postn expression in the epithelial tumor cells. Furthermore, using an in vitro model, we show a cross-regulation between FGFR, TGFβ and PI3K/AKT pathways to regulate Postn expression. In HER2-positive murine breast cancer cells, we found that basic FGF can repress Postn expression through a PKC-dependent pathway, while TGFβ can induce Postn expression in a SMAD-independent manner. Postn induction following the removal of the FGF-suppressive signal is dependent on PI3K/AKT signaling. Conclusion Overall, these results reveal a novel regulatory mechanism and shed light on how breast tumor cells acquire Postn expression. This complex regulation is likely to be cell type and cancer specific as well as have important therapeutic implications.

Smooth muscle-specific MMP17 (MT4-MMP) regulates the intestinal stem cell niche and regeneration after damage

Smooth muscle is an essential component of the intestine, both to maintain its structure and produce peristaltic and segmentation movements. However, very little is known about other putative roles that smooth muscle cells may have. Here, we show that smooth muscle cells may be the dominant suppliers of BMP antagonists, which are niche factors essential for intestinal stem cell maintenance. Furthermore, muscle-derived factors render epithelium reparative and fetal-like, which includes heightened YAP activity. Mechanistically, we find that the membrane-bound matrix metalloproteinase MMP17, which is exclusively expressed by smooth muscle cells, is required for intestinal epithelial repair after inflammation- or irradiation-induced injury. Furthermore, we propose that MMP17 affects intestinal epithelial reprogramming after damage indirectly by cleaving diffusible factor(s) such as the matricellular protein PERIOSTIN. Together, we identify an important signaling axis that establishes a role for smooth muscle cells as modulators of intestinal epithelial regeneration and the intestinal stem cell niche.