Vasopressin Receptor Type-2 Mediated Signaling in Renal Cell Carcinoma Stimulates Stromal Fibroblast Activation
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ABSTRACT:
SUBMITTER: Jamadar A
PROVIDER: S-EPMC9325308 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fibroblast growth factor (FGF)-23 induces hypertrophy and calcium (Ca2+) dysregulation in cardiomyocytes, leading to cardiac arrhythmia and heart failure. However, knowledge regarding the effects of FGF-23 on cardiac fibrogenesis remains limited. This study investigated whether FGF-23 modulates cardiac fibroblast activity and explored its underlying mechanisms. We performed MTS analysis, 5-ethynyl-2'-deoxyuridine assay, and wound-healing assay in cultured human atrial fibroblasts without and with FGF-23 (1, 5 and 25 ng/mL for 48 h) to analyze cell proliferation and migration. We found that FGF-23 (25 ng/mL, but not 1 or 5 ng/mL) increased proliferative and migratory abilities of human atrial fibroblasts. Compared to control cells, FGF-23 (25 ng/mL)-treated fibroblasts had a significantly higher Ca2+ entry and intracellular inositol 1,4,5-trisphosphate (IP3) level (assessed by fura-2 ratiometric Ca2+ imaging and enzyme-linked immunosorbent assay). Western blot analysis showed that FGF-23 (25 ng/mL)-treated cardiac fibroblasts had higher expression levels of calcium release-activated calcium channel protein 1 (Orai1) and transient receptor potential canonical (TRPC) 1 channel, but similar expression levels of α-smooth muscle actin, collagen type IA1, collagen type Ⅲ, stromal interaction molecule 1, TRPC 3, TRPC6 and phosphorylated-calcium/calmodulin-dependent protein kinase II when compared with control fibroblasts. In the presence of ethylene glycol tetra-acetic acid (a free Ca2+ chelator, 1 mM) or U73122 (an inhibitor of phospholipase C, 1 μM), control and FGF-23-treated fibroblasts exhibited similar proliferative and migratory abilities. Moreover, polymerase chain reaction analysis revealed that atrial fibroblasts abundantly expressed FGF receptor 1 but lacked expressions of FGF receptors 2-4. FGF-23 significantly increased the phosphorylation of FGF receptor 1. Treatment with PD166866 (an antagonist of FGF receptor 1, 1 μM) attenuated the effects of FGF-23 on cardiac fibroblast activity. In conclusion, FGF-23 may activate FGF receptor 1 and subsequently phospholipase C/IP3 signaling pathway, leading to an upregulation of Orai1 and/or TRPC1-mediated Ca2+ entry and thus enhancing human atrial fibroblast activity.
| S-EPMC8745152 | biostudies-literature
Project description:Carcinoma-associated fibroblasts (CAFs) play a key onco-supportive role during prostate cancer (PCa) development and progression. We previously reported that the reactive oxygen species (ROS)-producing enzyme NADPH oxidase 4 (Nox4) is essential for TGF?1-mediated activation of primary prostate human fibroblasts to a CAF-like phenotype. This study aimed to further investigate the functional relevance of prostatic Nox4 and determine whether pharmacological inhibition of stromal Nox4 abrogates paracrine-mediated PCa-relevant processes. RNA in situ hybridization revealed significantly elevated Nox4 mRNA levels predominantly in the peri-tumoral stroma of clinical PCa with intense stromal Nox4 staining adjacent to tumor foci expressing abundant TGF? protein levels. At pharmacologically relevant concentrations, the Nox1/Nox4 inhibitor GKT137831 attenuated ROS production, CAF-associated marker expression and migration of TGF?1-activated but not nonactivated primary human prostate fibroblasts. Similar effects were obtained upon shRNA-mediated silencing of Nox4 but not Nox1 indicating that GKT137831 primarily abrogates TGF?1-driven fibroblast activation via Nox4 inhibition. Moreover, inhibiting stromal Nox4 abrogated the enhanced proliferation and migration of PCa cell lines induced by TGF?1-activated prostate fibroblast conditioned media. These effects were not restricted to recombinant TGF?1 as conditioned media from PCa cell lines endogenously secreting high TGF?1 levels induced fibroblast activation in a stromal Nox4- and TGF? receptor-dependent manner. Importantly, GKT137831 also attenuated PCa cell-driven fibroblast activation. Collectively, these findings suggest the TGF?-Nox4 signaling axis is a key interface to dysregulated reciprocal stromal-epithelial interactions in PCa pathophysiology and provide a strong rationale for further investigating the applicability of Nox4 inhibition as a stromal-targeted approach to complement current PCa treatment modalities.
| S-EPMC6067067 | biostudies-literature
Project description:Arginine vasopressin (AVP) and its type-2 receptor (V2R) play an essential role in the regulation of salt and water homeostasis by the kidneys. V2R activation also stimulates proliferation of renal cell carcinoma (RCC) cell lines in vitro. The current studies investigated V2R expression and activity in human RCC tumors, and its role in RCC tumor growth. Examination of the cancer genome atlas (TCGA) database, and analysis of human RCC tumor tissue microarrays, cDNA arrays and tumor biopsy samples demonstrated V2R expression and activity in clear cell RCC (ccRCC). In vitro, V2R antagonists OPC31260 and Tolvaptan, or V2R gene silencing reduced wound closure and cell viability of 786-O and Caki-1 human ccRCC cell lines. Similarly in mouse xenograft models, Tolvaptan and OPC31260 decreased RCC tumor growth by reducing cell proliferation and angiogenesis, while increasing apoptosis. In contrast, the V2R agonist dDAVP significantly increased tumor growth. High intracellular cAMP levels and ERK1/2 activation were observed in human ccRCC tumors. In mouse tumors and Caki-1 cells, V2R agonists reduced cAMP and ERK1/2 activation, while dDAVP treatment had the reverse effect. V2R gene silencing in Caki-1 cells also reduced cAMP and ERK1/2 activation. These results provide novel evidence for a pathogenic role of V2R signaling in ccRCC, and suggest that inhibitors of the AVP-V2R pathway, including the FDA-approved drug Tolvaptan, could be utilized as novel ccRCC therapeutics.
| S-EPMC7007354 | biostudies-literature
Project description:Excessive mitochondrial fission acts as a pro-proliferative marker in some cancers and organ fibrosis; its potential role in renal fibroblast activation and fibrogenesis has never been investigated. Here, we showed more pronounced fragmented mitochondria in fibrotic than in non-fibrotic renal fibroblast in humans and mice. In a mouse model of obstructive nephropathy, phosphorylation of Drp1 at serine 616 (p-Drp1S616) and acetylation of H3K27(H3K27ac) was increased in fibrotic kidneys; pharmacological inhibition of mitochondrial fission by mdivi-1 substantially reduced H3K27ac levels, fibroblasts accumulation, and interstitial fibrosis. Moreover, mdivi-1 treatment was able to attenuate the established renal fibrosis. In cultured renal interstitial fibroblasts, targeting Drp1 using pharmacological inhibitor or siRNA suppressed TGF-?1-elicited cell activation and proliferation, as evidenced by inhibiting expression of ?-smooth muscle actin (?-SMA) and collagen I, as well as by reducing DNA synthesis. In contrast, Drp1 deletion enhanced cell apoptosis, along with decreased mitochondrial fragmentation, mtROS elevation, and glycolytic shift upon TGF-?1 stimulation. In Drp1 deletion fibroblasts, re-expression of wild-type Drp1 rather than Drp1S616A mutant restores the reduction of TGF-?-induced-Drp1 phosphorylation, H3K27ac, and cell activation. Moreover, TGF-?1 treatment increased the enrichment of H3K27ac at the promoters of ?-SMA and PCNA, which was reversed in Drp1-knockdown fibroblasts co-transfected with empty vector or Drp1S616A, but not wild-type Drp1. Collectively, our results imply that inhibiting p-Drp1S616-mediated mitochondrial fission attenuates fibroblast activation and proliferation in renal fibrosis through epigenetic regulation of fibrosis-related genes transcription and may serve as a therapeutic target for retarding progression of chronic kidney disease.
| S-EPMC6965618 | biostudies-literature
Project description:We generated VEGF-null fibrosarcomas from VEGF-loxP mouse embryonic fibroblasts to investigate the mechanisms of tumor escape after VEGF inactivation. These cells were found to be tumorigenic and angiogenic in vivo in spite of the absence of tumor-derived VEGF. However, VEGF derived from host stroma was readily detected in the tumor mass and treatment with a newly developed anti-VEGF monoclonal antibody substantially inhibited tumor growth. The functional significance of stroma-derived VEGF indicates that the recruitment of stromal cells is critical for the angiogenic and tumorigenic properties of these cells. Here we identified PDGF AA as the major stromal fibroblast chemotactic factor produced by tumor cells, and demonstrated that disrupting the paracrine PDGFR alpha signaling between tumor cells and stromal fibroblasts by soluble PDGFR alpha-IgG significantly reduced tumor growth. Thus, PDGFR alpha signaling is required for the recruitment of VEGF-producing stromal fibroblasts for tumor angiogenesis and growth. Our findings highlight a novel aspect of PDGFR alpha signaling in tumorigenesis.
| S-EPMC514949 | biostudies-literature
Project description:Vasopressin regulates renal water excretion by binding to a Gα s-coupled receptor (V2R) in collecting duct cells, resulting in increased water permeability through regulation of the aquaporin-2 (AQP2) water channel. This action is widely accepted to be associated with cAMP-mediated activation of protein kinase A (PKA). Here, we use phosphoproteomics in collecting duct cells in which PKA has been deleted (CRISPR-Cas9) to identify PKA-independent responses to vasopressin. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Upregulated sites in PKA-null cells include Ser256 of AQP2, which is critical to regulation of AQP2 trafficking. In addition, phosphorylation changes in the protein kinases Stk39 (SPAK) and Prkci (an atypical PKC) are consistent with PKA-independent regulation of these protein kinases. Target motif analysis of the phosphopeptides increased in PKA-null cells indicates that vasopressin activates one or more members of the AMPK/SNF1-subfamily of basophilic protein kinases. In vitro phosphorylation assays using recombinant, purified SNF1-subfamily kinases confirmed postulated target specificities. Of interest, measured IBMX-dependent cAMP levels were an order of magnitude higher in PKA-null than in PKA-intact cells, indicative of a PKA-dependent feedback mechanism. Overall, the findings support the conclusion that V2-receptor mediated signaling in collecting duct cells is in part PKA-independent.
| S-EPMC9200475 | biostudies-literature
Project description:Cancer-associated fibroblasts (CAFs) play significant roles in drug resistance through different ways. Antitumor therapies, including molecular targeted interventions, not only effect tumor cells but also modulate the phenotype and characteristics of CAFs, which can in turn blunt the therapeutic response. Little is known about how stromal fibroblasts respond to poly (ADP-ribose) polymerase inhibitors (PARPis) in ovarian cancer (OC) and subsequent effects on tumor cells. This is a study to evaluate how CAFs react to PARPis and their potential influence on PARPi resistance in OC. We discovered that OC stromal fibroblasts exhibited intrinsic resistance to PARPis and were further activated after the administration of PARPis. PARPi-challenged fibroblasts displayed a specific secretory profile characterized by increased secretion of CCL5, MIP-3α, MCP3, CCL11, and ENA-78. Mechanistically, increased secretion of CCL5 through activation of the NF-κB signaling pathway was required for PARPi-induced stromal fibroblast activation in an autocrine manner. Moreover, neutralizing CCL5 partly reversed PARPi-induced fibroblast activation and boosted the tumor inhibitory effect of PARPis in both BRCA1/2-mutant and BRCA1/2-wild type xenograft models. Our study revealed that PARPis could maintain and improve stromal fibroblast activation involving CCL5 autocrine upregulation. Targeting CCL5 might offer a new treatment modality in overcoming the reality of PARPi resistance in OC.
| S-EPMC8190269 | biostudies-literature
Project description:RationaleSmall molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown.ObjectiveWe sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart.Methods and resultsRNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-β (transforming growth factor-β) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-β-mediated cardiac fibroblast activation.ConclusionsThese findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4. The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.
| S-EPMC7310347 | biostudies-literature
Project description:Enhanced arginine vasopressin levels are associated with increased mortality during end-stage human heart failure, and cardiac arginine vasopressin type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased ?-adrenergic receptor (?AR) responsiveness. This led us to hypothesize that V1AR signaling regulates ?AR responsiveness and in doing so contributes to development of heart failure.Transaortic constriction resulted in decreased cardiac function and ?AR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased ?AR ligand affinity, as well as ?AR-induced Ca(2+) mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of ?AR responsiveness was demonstrated to occur in a previously unrecognized Gq protein-independent/G protein receptor kinase-dependent manner.This newly discovered relationship between cardiac V1AR and ?AR may be informative for the treatment of patients with acute decompensated heart failure and elevated arginine vasopressin.
| S-EPMC4229385 | biostudies-literature
Project description:BackgroundRenal cell carcinoma (RCC) is a clinically common tumor in the urinary system, showing an upward trend of both incidence and mortality. Apolipoprotein C1 (APOC1) has been identified as a vital regulator in tumor progression. This study aims to uncover the biological function of APOC1 in RCC process and the underlying mechanism.MethodsDifferential levels of APOC1 in RCC samples and normal tissues in a downloaded TCGA profile and clinical samples collected in our center were detected by quantitative reverse transcription PCR (qRT-PCR). The prognostic value of APOC1 in RCC was assessed by depicting Kaplan-Meier survival curves. After intervening APOC1 level by transfection of sh-APOC1 or oe-APOC1, changes in phenotypes of RCC cells were examined through CCK-8, colony formation, Transwell assay and flow cytometry. Subsequently, protein levels of EMT-related genes influenced by APOC1 were determined by Western blot. The involvement of the Wnt3a signaling in APOC1-regulated malignant process of RCC was then examined through a series of rescue experiments. Finally, a RCC xenograft model was generated in nude mice, aiming to further clarify the in vivo function of APOC1 in RCC process.ResultsAPOC1 was upregulated in RCC samples. Notably, its level was correlated to overall survival of RCC patients, displaying a certain prognostic value. APOC1 was able to stimulate proliferative, migratory and invasive abilities in RCC cells. The Wnt3a signaling was identified to be involved in APOC1-mediated RCC process. Notably, Wnt3a was able to reverse the regulatory effects of APOC1 on RCC cell phenotypes. In vivo knockdown of APOC1 in xenografted nude mice slowed down the growth of RCC.ConclusionsAPOC1 stimulates the malignant process of RCC via targeting the Wnt3a signaling.
| S-EPMC7802262 | biostudies-literature