mTOR, the target of rapamycin, has been promoted as a potential target for cancers, transplantations and even lung fibrosis. However, paradoxically, targeting mTOR has been reported to result in profibrotic side effects. Some recent publications highlight why this might be.

In this report, chairs of the 7th International Workshop on the CCN family of Genes, review the progress made in understanding the biological functions of CCN proteins (CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6) with a particular focus on their implications in various pathological conditions, including cancer, fibrosis, diabetes, and cardiovascular diseases.

Vascular smooth muscle cells (VSMCs) are subject to changing hemodynamic stimuli that alter cytoskeletal dynamics, cellular architecture, and structure-associated signal transduction. Tensional stress, force application, and structural perturbations are sensed by VSMCs and impact the physiological as well as pathophysiological responses of the vasculature. Microtubule-targeting drugs provide useful tools to analyze cytoskeletal-associated signaling pathways and their linkages to pathological outcomes. Architecture-based controls on a subset of profibrotic genes commonly expressed in vascular disease are highlighted by their frequent induction in mechanically manipulated cells and with associated changes in cytoskeletal dynamics. VSMCs respond to biomechanical cues by activating several kinase cascades, leading to gene reprogramming. It is apparent that a significant fraction of the vast repertoire of signaling intermediates, moreover, are sequestered on the cytoskeletal framework in an "inactive state." Reorganization within these networks due to fluctuating mechanical forces could release these effectors from their cytoskeletal anchors, thus alleviating the "repressive state" resulting in downstream signaling. Indeed, recent findings indicate that microtubule disruption in VSMCs rapidly stimulates pp60c-src kinase activation and epidermal growth factor receptor (EGFR) transphosphorylation at Y845, a src kinase target residue. EGFR genetic deficiency, pharmacological inhibition of EGFR signaling, or adenoviral delivery of the kinase-deficient EGFRK721A construct effectively blocked colchicine-stimulated expression of two prominent vascular profibrotic genes, plasminogen activator inhibitor type-1 (PAI-1; SERPINE1) and connective tissue growth factor (CTGF; CCN2). Signaling intermediates involved in microtubule collapse-initiated PAI-1/CTGF induction in VSMCs include the MEK/ERK, Rho/ROCK, and SMAD2/3 pathways. This review highlights commonalities and differences in signaling events that facilitate expression of vascular disease-relevant genes initiated as a consequence of loss of microtubular integrity.

The Hippo pathway has been associated with regulation of early follicle growth. Studies of murine ovaries suggest that changes in the actin cytoskeleton, caused by fragmentation, result in inhibition of the Hippo pathway, and in turn, may activate follicle growth. In humans, the connections between fragmentation, the actin cytoskeleton, and follicle activation are yet to be confirmed. In this study, we investigated the impact in vitro fragmentation of a human ovarian cortex on (a) actin polymerization, (b) components of the Hippo pathway, and (c) follicle growth in vivo. The results showed that the ratio between globular and filamentous actin remained unchanged at all timepoints (0, 10, 30, 60, 120, and 240 min) following tissue fragmentation. Neither was the Hippo pathway effector protein YES-associated protein upregulated nor was gene expression of the downstream growth factors CCN2, CCN3, or CCN5 increased at any timepoint in the fragmented cortex. Furthermore, the number of growing follicles was similar in fragmented and intact cortex pieces after 6 weeks' xenotransplantation. However, the total number of surviving follicles was considerably lower in the fragmented cortex compared with intact tissue, suggesting detrimental effects of fragmentation on tissue grafting. These results indicate that fragmentation is likely to be ineffective to activate follicle growth in the human ovarian cortex.

Keywords: Hippo pathway; actin; female infertility; follicle activation; ovarian cortex.

Growth-plate chondrocytes undergo proliferation, maturation, hypertrophic differentiation, and calcification; and these processes can be reproduced in vitro in a chondrocyte culture system. Using this system, we have shown that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes all stages of proliferation, maturation, hypertrophic differentiation, and calcification, thus suggesting that CCN2 is a multifunctional growth factor for chondrocytes and plays important roles in chondrocyte proliferation and differentiation. In this chapter, we describe how to evaluate CCN2 functions in these processes occurring in cultured chondrocytes. Evaluation strategies for cell proliferation include measuring DNA synthesis by [3H]-thymidine incorporation, cellular metabolic activity, and cell number with a hemocytometer. Next, evaluation strategies to assess maturation are analysis of the gene expression of markers of mature chondrocytes, and examination of proteoglycan and collagen synthesis by using radioactive compounds. In addition, cytohistochemical detection of glycosaminoglycans (GAGs), such as chondroitin sulfate, by use of alcian blue and toluidine blue staining is useful to evaluate chondrocyte maturation. These methods can be also used for evaluation of physiological functions of CCN2 in permanent chondrocytes such as articular and auricular chondrocytes, which do not calcify under physiological conditions. Next, evaluation of hypertrophic differentiation is performed by detecting type X collagen, which is specific marker of hypertrophic chondrocytes, and by measuring alkaline phosphatase (ALP) activity. Finally, evaluation of calcification is performed by detecting matrix calcification by use of alizarin red staining and by examining the incorporation of 45Ca into cartilaginous matrix. These methods would be useful for the evaluation not only of CCN2 but also of its derivatives and other CCN proteins.

Transforming growth factor beta (TGFβ) a multifunctional cytokine is known to regulate cell proliferation, differentiation, migration and survival. Although there is variable expression of modulators of TGFβ action during differentiation, a differential effect on fat cell metabolism at different stages of adipocyte differentiation was unclear. In this study, 3T3L1 cells were used as an in vitro model to study the effect of TGFβ on adipogenic and thermogenic markers at various stages of preadipocyte to mature adipocyte differentiation. As in our earlier studies [5] on the effect of TGFβ on CEBP's we used a standard differentiation mix, and one with the addition of rosiglitazon. RhTGFβ1 was added to undifferentiated adipocytes (preadipocytes) and to adipocytes at day 0 (commitment stage) as well as day 10 (terminal differentiation). Cellular responses in terms of Pref1, PPARγ, TLE3, PGC1a, PRDM16, UCP1 and UCP2 mRNA levels and selected protein products, were determined. Increases in PPARγ PRDM16, UCP1 and UCP2 mRNA and decreases in Pref1 are good indicators of successful differentiation. The early addition of rhTGFβ1 during commitment decreased PPARγ, PRDM16, TLE3, UCP1 and UCP2 mRNA and decreased PRDM16 protein consistent with our earlier report on the inhibition of CEBP's by TGFβ and CCN2. The addition of rhTGFβ1 on mature adipocyte at day 10 increased UCP1 mRNA and increased PRDM16 and UCP1 proteins. In this recent study our results suggest that TGFβ1 added late enhances the thermogenic potential of mature cells and causes 3T3L1 cells to differentiate to resemble brown or beige rather than white adipose tissue.

CCN1 and CCN2 are members of the CCN family and play essential roles in the regulation of multiple female reproductive functions, including ovulation. Cyclooxygenase-2 (COX2) is a critical mediator of ovulation and can be induced by sphingosine-1-phosphate (S1P) through the S1P_1/3-mediated Yes-associated protein (YAP) signaling. However, it is unclear whether CCN1 or CCN2 can mediate S1P-induced upregulation of COX2 expression and increase in prostaglandin E2 (PGE2) production in human granulosa-lutein (hGL) cells. In the present study, we investigated the effects of S1P on the expressions of CCN1 and CCN2 in hGL cells. Additionally, we used a dual inhibition approach (siRNA-mediated silencing and small molecular inhibitors) to investigate the molecular mechanisms of S1P effects. Our results showed that S1P treatment significantly upregulated the expression of CCN1 and CCN2 in a concentration-dependent manner in hGL cells. Additionally, inhibition or silencing of S1P₁, but not S1P₃, completely abolished the S1P-induced upregulation of CCN2 expression. Furthermore, we demonstrated that S1P-induced nuclear translocation of YAP and inhibition or silencing of YAP completely abolished the S1P-induced upregulation of CCN1 and CCN2 expression. Notably, silencing of CCN2, but not CCN1, completely reversed the S1P-induced upregulation of COX2 expression and the increase in PGE2 production. Thus, CCN2 mediates the S1P-induced upregulation of COX2 expression through the S1P₁-mediated signaling pathway in hGL cells. Our findings expand our understanding of the molecular mechanism underlying the S1P-mediated cellular activities in the human ovary.

CCN2/CTGF is a matricellular protein that is known to enhance transforming growth factor-β signaling and to induce a myofibroblast-like phenotype in a variety of cell types. Here, we investigated Ccn2/Ctgf promotor activity during development and in the adult mouse eye, using CTGF^LacZ/+ mice in which the β-galactosidase reporter gene LacZ had been inserted into the open reading frame of Ccn2/Ctgf. Promotor activity was assessed by staining for β-galactosidase activity and by immunolabeling using antibodies against β-galactosidase. Co-immunostaining using antibodies against glutamine synthetase, glial fibrillary acidic protein, choline acetyltransferase, and CD31 was applied to identify specific cell types. Ccn2/Ctgf promotor activity was intense in neural crest-derived cells differentiating to corneal stroma and endothelium, and to the stroma of choroid, iris, ciliary body, and the trabecular meshwork during development. In the adult eye, a persistent and very strong promotor activity was present in the trabecular meshwork outflow pathways. In addition, endothelial cells of Schlemm's canal, and of retinal and choroidal vessels, retinal astrocytes, Müller glia, and starburst amacrine cells were stained. Very strong promoter activity was seen in the astrocytes of the glial lamina at the optic nerve head. We conclude that CCN2/CTGF signaling is involved in the processes that govern neural crest morphogenesis during ocular development. In the adult eye, CCN2/CTGF likely plays an important role for the trabecular meshwork outflow pathways and the glial lamina of the optic nerve head.

Keywords: Astrocyte; Eye development; Glial lamina; Neural crest; Trabecular meshwork.

G protein-coupled estrogen receptor 1 (GPER1) is a potential therapeutic target for treating triple-negative breast cancers (TNBC). However, modulators for GPER1 that can be used to treat TNBC have not appeared. Berberine (BBR) is a bioactive isoquinoline alkaloid with high oral safety. In recent years, BBR has shown an inhibitory effect on TNBC tumors such as MDA-MB-231, but the molecular target remains unclear, which hinders related clinical research. Our work proved that BBR is a modulator of GPER1 that can inhibit cell viability, migration, and autophagy of MDA-MB-231 cells. The inhibitory effect of BBR on MDA-MB-231 cells has a dependence on estrogen levels. Although BBR promoted the proteasome, which is a major factor in the degradation of GPER1, it could still induce the protein level of GPER1. Correspondingly, the transcription of cellular communication network factor 2 (CCN2) was promoted. BBR could bind to GPER1 directly and change the secondary structure of GPER1, as in the case of 17β-estradiol (E2). In addition, BBR induced not only a high degree of co-localization of GPER1 and microtubule-associated protein 1 light chain 3 (MAP1LC3), but also the accumulation of sequestosome 1 (SQSTM1/p62) by the inhibition of the nuclear translocation of the nuclear factor-kappa B (NF-κB) subunit (RELA/p65), which indicates NF-κB inhibition and anti-cancer effects. This result proved that the promotional effect of BBR on the GPER1/NF-κB pathway was closely related to its inhibitory effect on autophagy, which may serve as a new mechanism by which to explain the inhibitory effect of BBR on MDA-MB-231 cells and expand our understanding of the function of both BBR and GPER1.

Keywords: GPER1; MAP1LC3; NF-κB; berberine; binding; co-localization.

Connective tissue growth factor (CTGF), also known as CCN2, is a member of the CCN protein family of secreted proteins with roles in diverse biological processes. CTGF regulates biological functions such as cell proliferation, migration, adhesion, wound healing, and angiogenesis. In this study, we demonstrate a mechanistic link between CTGF and enhanced aerobic glycolysis in triple-negative breast cancer (TNBC). We found that CTGF is overexpressed in TNBC and high CTGF expression is correlated with a poor prognosis. Also, CTGF was required for in vivo tumorigenesis and in vitro proliferation, migration, invasion, and adhesion of TNBC cells. Our results indicate that extracellular CTGF binds directly to integrin αvβ3, activating the FAK/Src/NF-κB p65 signaling axis, which results in transcriptional upregulation of Glut3. Neutralization of CTGF decreased cell proliferation, migration, and invasion through downregulation of Glut3-mediated glycolytic phenotypes. Overall, our work suggests a novel function for CTGF as a modulator of cancer metabolism, indicating that CTGF is a potential therapeutic target in TNBC.

Acquisition of endometrial receptivity for embryo implantation is one of the crucial processes during pregnancy and is induced mainly by progesterone and enhanced by conceptus signals. Prokineticin 1 (PROK1) is characterized as a secretory protein with diverse functions in various tissues, including the reproductive tract. PROK1, with its receptor PROKR1, are up-regulated in the porcine endometrium during implantation and in women's receptive endometrium and decidua. However, the function of PROK1 in embryo-maternal communication has still not been fully elucidated. Hence, we hypothesize that PROK1 is involved in endometrial receptivity development and implantation in pigs. In this study, using the porcine in vivo model of intrauterine infusions of estradiol-17β (E2) and prostaglandin E2 (PGE2), we revealed that these hormones elevated endometrial expression of PROK1 and PROKR1 mRNA, respectively. Moreover, E2, acting synergistically with PGE2, increased PROKR1 protein expression. We also evidenced that PROK1-PROKR1 signaling induced expression of following genes and/or proteins CCN2, CDH13, FGF2, NFATC2, ANGPT1, ANGPT2, CDH1, MUC4, SPP1, IFNG, IL6, LIF, LIFR, TNF, TGFB3, and FGF9, as well as phosphorylation of PTK2 and secretion of IL6 and IL11 by endometrial explants in vitro. Ingenuity pathway analysis revealed that functions associated with the PROK1-regulated genes/proteins include cell-to-cell contact, cell attachment, migration and viability, differentiation of epithelial tissue, leukocyte migration, inflammatory response, angiogenesis, and vasculogenesis. Summarizing, our study suggests that PROK1 acts pleiotropically as an embryonic signal mediator that regulates endometrial receptivity by increasing the expression of the genes and proteins involved in implantation and pregnancy establishment in pigs.

Keywords: endometrial receptivity; implantation; pregnancy; prokineticin 1; prokineticin receptor 1; the pig.

Aims: Excessive inflammatory signaling and pathological remodeling of the extracellular matrix drive cardiac fibrosis and require changes in gene expression. Materials and methods: Using bioinformatics, both tissue-specific expression profiles and epigenomic profiles of some genes critical for cardiac fibrosis were examined, namely, NLRP3, MMP2, MMP9, CCN2/CTGF, AGT (encodes angiotensin II precursors) and hsa-mir-223 (post-transcriptionally regulates NLRP3). Results: In monocytes, neutrophils, fibroblasts, venous cells, liver and brain, enhancers or super-enhancers were found that correlate with high expression of these genes. One enhancer extended into a silent gene neighbor. These enhancers harbored tissue-specific foci of DNA hypomethylation, open chromatin and transcription factor binding. Conclusions: This study identified previously undescribed enhancers containing hypomethylated transcription factor binding subregions that are predicted to regulate expression of these cardiac fibrosis-inducing genes.

Keywords: DNA methylation; DNase I hypersensitive sites; cardiovascular disease; enhancers; epigenetics; fibrosis; inflammasomes; lincRNA; super-enhancers; transcription factor binding.

Purpose/Aim: Volumetric muscle loss (VML) is a devastating orthopaedic injury resulting in chronic persistent functional deficits, loss of joint range of motion, pathologic fibrotic deposition and lifelong disability. However, there is only limited mechanistic understanding of VML-induced fibrosis. Herein we examined the temporal changes in the fibrotic deposition at 3, 7, 14, 28, and 48 days post-VML injury. Materials and Methods: Adult male Lewis rats (n=39) underwent a full thickness ~20% (~85mg) VML injury to the tibialis anterior (TA) muscle unilaterally, the contralateral TA muscle served as the control group. All TA muscles were harvested for biochemical and histologic evaluation. Results: The ratio of collagen I/III was decreased at 3, 7, and 14 days post-VML, but significantly increased at 48 days. Decorin content followed an opposite trend, significantly increasing by day 3 before dropping to below control levels by 48 days. Histological evaluation of the defect area indicates a shift from loosely packed collagen at early time points post-VML, to a densely packed fibrotic scar by 48 days. Conclusions: The shift from early wound healing efforts to a fibrotic scar with densely packed collagen within the skeletal muscle occurs around 21 days after VML injury through dogmatic synchronous reduction of collagen III and increase in collagen I. Thus, there appears to be an early window for therapeutic intervention to prevent pathologic fibrous tissue formation, potentially by targeting CCN2/CTGF or using decorin as a therapeutic.

Keywords: Collagen; Fibrosis; Neuromusculoskeletal injury; Orthopaedic trauma; Skeletal muscle injury.

Fibrosis is perpetuated by an autocrine, pro-adhesive signaling loop maintained by the synthetic and contractile abilities of myofibroblasts and the stiff, highly-crosslinked extracellular matrix. Transcriptional complexes that are exquisitely responsive to mechanotransduction include the co-activator YAP1, which regulates the expression of members of the CCN family of matricellular proteins such as CCN2 and CCN1. Although selective YAP1 inhibitors exist, the effect of these inhibitors on profibrotic gene expression in fibroblasts is largely unknown, and is the subject of our current study. Herein, we use genome-wide expression profiling, real-time polymerase chain reaction and Western blot analyses, cell migration and collagen gel contraction assays to assess the ability of a selective YAP inhibitor verteporfin (VP) to block fibrogenic activities in dermal fibroblasts from healthy individual human controls and those from isolated from fibrotic lesions of patients with diffuse cutaneous systemic sclerosis (dcSSc). In control fibroblasts, VP selectively reduced expression of fibrogenic genes and also blocked the ability of TGFbeta to induce actin stress fibers in dermal fibroblasts. VP also reduced the persistent profibrotic phenotype of dermal fibroblasts cultured from fibrotic lesions of patients with dcSSc. Our results are consistent with the notion that, in the future, YAP1 inhibitors may represent a novel, valuable method of treating fibrosis as seen in dcSSc.

Keywords: CCN2; CTGF; Connective tissue growth factor; Fibrosis; Mechanotransduction; Myofibroblasr; Scleroderma; Verteporfin; YAP1.

Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities.

Keywords: CCN2; Cartilage; Chondrocytes; FGF-1; Osteoarthritis.

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix proteins and myofibroblast differentiation. Our previous findings have implicated resistin in cardiac fibrosis; however, the molecular mechanisms underlying this process are still unclear. Here we investigated the role of resistin in fibroblast-to-myofibroblast differentiation and elucidated the pathways involved in this process. Fibroblast-to-myofibroblast transdifferentiation was induced with resistin or TGFβ1 in NIH-3T3 and adult cardiac fibroblasts. mRNA and protein expression of fibrotic markers were analyzed by qPCR and immunoblotting. Resistin-knockout mice, challenged with a high-fat diet (HFD) for 20 weeks to stimulate cardiac impairment, were analyzed for cardiac function and fibrosis using histologic and molecular methods. Cardiac fibroblasts stimulated with resistin displayed increased fibroblast-to-myofibroblast conversion, with increased levels of αSma, col1a1, Fn, Ccn2 and Mmp9, with remarkable differences in the actin network appearance. Mechanistically, resistin promotes fibroblast-to-myofibroblast transdifferentiation and fibrogenesis via JAK2/STAT3 and JNK/c-Jun signaling pathways, independent of TGFβ1. Resistin-null mice challenged with HFD showed an improvement in cardiac function and a decrease in tissue fibrosis and reduced mRNA levels of fibrogenic markers. These findings are the first to delineate the role of resistin in the process of cardiac fibroblast-to-myofibroblast differentiation via JAK/STAT3 and JNK/c-Jun pathways, potentially leading to stimulation of cardiac fibrosis.

Keywords: Fibroblast/Myofibroblast differentiation; JAK2/STAT3; JNK/c-Jun; Myocardial Fibrosis; Resistin; SP600125: JNK inhibitor; WP1066: JAK2 inhibitor.

Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated "M10," with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFβ-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor (CTGF, CCN2) and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients.

Following publication of their article "CCN2 inhibits lung cancer metastasis through promoting DAPK-dependent anoikis and inducing EGFR degradation", the authors reported an error in Fig.6b. α-Tubulin image of rCCN2 treatment (upper panel in CL1-5) only showed eight lanes, when there should be nine.

Human gingival overgrowth may occur as a side effect of chronic administration of some therapeutic agents. The mechanisms responsible for the gingival tissues lesions, fibrosis and inflamation, involve an impaired balance between the production and the degradation of type I collagen. It has been demonstrated that CCN2/CTGF, a connective tissue growth factor, is highly expressed in the gingival tissues and positively correlated with the degree of fibrosis in the drug-induced gingival overgrowth. The aim of this study was to identify the presence and localization of CCN2/CTGF and CCN1/Cyr61, members of the same molecular family, in gingival tissues of cyclosporin A- and nifedipine-treated rats, by immunohistochemistry. Staining was evaluated with light microscope and the results show cellular and extracellular CTGF in nifedipin gingival overgrowth tissues with intensity of labeling higher compared to the CsA gingival overgrowth tissues or the controls. The staining for Cyr61 shows its intracellular localization with no diference of labeling intensity between drug-induced gingival overgrowth and normal tissues. Also, we were interested in the gingival TGF-â expression in those animals. We didn't find any commercial anti-rat TGF antibody and our anti-human antibody shows no cross-reactivity with rat tissues. The data from our study sustain the involvement of CTGF and Cyr61 as growth factors in the gingival tissues and the CTGF association with drug-induced gingival overgrowth.

Skin is composed of multiple layers, including the epidermis, dermis, and hypodermis. Although several biological activities of fisetin have been reported, beneficial effects and the functions of fisetin in skin remain unclear. B16F10 melanoma cells, human skin fibroblasts, and 3T3-L1 cells were used to examine the beneficial effects of fisetin in skin health. α-MSH- and IBMX-induced melanosis in B16F10 melanoma cells was inhibited by fisetin treatment, which also enhanced mRNA expression levels of skin fibril-related genes via the CCN2/TGF-β signaling pathway. Decreased intracellular lipid accumulation via down-regulation of transcriptional factors through activation of the CCN2/TGF-β signaling pathway was observed. A novel function of fisetin in skin health via down-regulation of melanosis and adipogenesis, and up-regulation of skin fibril-related genes was observed. Evidence for development of nutri-cosmetics for skin health is presented.

Background. The aim of this study was to assess the molecular effect of ulipristal acetate (UPA) on gene expression in myometrium and endometrium of patients with symptomatic fibroids.Research design and methods: Tissues isolated from four women treated preoperatively with UPA (5 mg) were compared to those from untreated controls using Nanostring platform to assess the expression of 75 candidate genes modulated by UPA and ovarian steroids. Deregulated genes were then validated by real-time PCR.Results: In myometrium UPA exerted an antagonistic effect similar to that observed in fibroids. In UPA-treated endometrium, six genes were identified as highly and significantly upregulated, including matricellular genes CCN1(54-fold, p = 0.0018) and CCN2 (11-fold, p = 0.00044), Krüppel-like factor 4 (KLF4, >3-fold, p = 0.0036) and mast cell markers including tryptases TPSAB1/TPSB2 (31-fold, p = 0.023) and carboxypeptidase A (CPA3, 17-fold, p = 0.05).Conclusions: In endometrium, UPA induced the expression of genes involved in fibrogenesis and mast cell function, some of them being widely involved in hepatic injury, which could explain the marked fibrosis and inflammatory cell infiltration observed in explanted livers from patients under UPA treatment.

Keywords: Endometrium; Nanostring platform; fibroids; fibrosis; myometrium; qPCR; ulipristal acetate.

Idiopathic subglottic stenosis (iSGS) is a progressive fibrotic disease characterized by life-threatening airway narrowing. While the molecular underpinnings are unknown, our previous reports showing that subglottic Serial Intra-Lesional Steroid Injections (SILSI) improves clinical outcomes suggests a steroid-sensitive pathway in iSGS. We conducted a prospective study to determine the changes in profibrotic markers during SILSI to identify steroid-sensitive profibrotic drivers. Seven newly diagnosed iSGS patients were recruited for SILSI. Subglottic biopsies before and after SILSI treatments were evaluated for histological and molecular markers by confocal microscopy and RT-qPCR. At baseline, iSGS subglottis contained abundant vimentin+/αSMA- fibroblasts, intermingled with a matrix of fibronectin, and type I and VI collagen. TGFβ1 was upregulated primarily in glandular epithelium. CCN2 was mainly upregulated in stromal fibroblasts surrounding TGFβ1+ glandular structures. SILSI improved iSGS by reducing fibroblast infiltration and increasing matrix remodeling. Mechanistically, SILSI counteracted the effects of TGFβ1 by inducing MMP9 expression while repressing CCN2 expression, without affecting TGFβ1 levels. Treatment of primary iSGS-derived fibroblasts with TGFβ1 recapitulated aspects of the disease in-vivo, demonstrating that the induction in CCN2 and repression of MMP9 are caused by changes in histone acetylation induced by TGFβ1. Triamcinolone counteracted the co-regulation of these genes by impairing SMAD2/3 binding to promoter regions, and not through histone acetylation. In conclusion, SILSI counteracts a dysregulated TGFβ1/CCN2/MMP9 axis involved in iSGS development.

Keywords: CCN2; MMP9; TGFβ1; airway; fibrosis; idiopathic fibrosis; idiopathic subglottic stenosis; serial intra-lesion steroid injections; steroids; triamcinolone.

Thanks to many advances in genetic manipulation, mouse models have become very powerful in their ability to interrogate biological processes. In order to precisely target expression of a gene of interest to particular cell types, intersectional genetic approaches utilizing two promoter/enhancers unique to a cell type are ideal. Within these methodologies, variants that add temporal control of gene expression are the most powerful. We describe the development, validation and application of an intersectional approach that involves three transgenes, requiring the intersection of two promoter/enhancers to target gene expression to precise cell types. Furthermore, the approach utilizes available lines expressing tTA/rTA to control timing of gene expression based on whether doxycycline is absent or present, respectively. We also show that the approach can be extended to other animal models, using chicken embryos. We generated three mouse lines targeted at the Tigre (Igs7) locus with TRE-loxP-tdTomato-loxP upstream of three genes (p21, DTA and Ctgf) and combined them with Cre and tTA/rtTA lines that target expression to the cerebellum and limbs. Our tools will facilitate unraveling biological questions in multiple fields and organisms.

Keywords: Ccn2/Ctgf; Cre; DRAGON allele; DTA; Diphtheria toxin; Inducible and reversible gene misexpression; Intersectional genetics; P21; RtTA; TTA; Tigre locus; Tissue-specific; Tox176.

In the kidney glucose is filtered by the glomerulus and reabsorbed by sodium glucose cotransporter 2 (SGLT2) in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological changes seen in Diabetic Kidney Disease (DKD) in response to elevated glucose. We developed a model of DKD using primary human PTECs with exposure to high D-glucose and TGF-β1 and propose a role for SGLT2 inhibition in regulating fibrosis. Western blotting was performed to detect cellular and secreted proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on D-glucose, 25mM, and TGF-β1, 0.75ng/ml, treated cells was explored using dapagliflozin and U0126, respectively. Only the combination of high D-glucose and TGF-β1 treatment significantly upregulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-β1 increased cellular phosphoSSXS-Smad3 serines 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-β1; this was significantly reduced by SGLT2 and MEK inhibition. We show that high D-glucose and TGF-β1 are both required for CCN2 expression. This treatment also caused Smad3 linker phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 -ERK mediated promotion of TGF-β1/Smad3 signalling inducing pro-fibrotic growth factor secretion. Our data evinces support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney.

Keywords: Dapagliflozin; Diabetic Kidney Disease; Fibrosis; Glucose; Glucose Transporters; Smad3.