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The platelet-derived growth factor receptor/STAT3 signaling pathway regulates the phenotypic transition of corpus cavernosum smooth muscle in rats.

ABSTRACT: Erectile dysfunction (ED) is a common clinical disease that is difficult to treat. We previously found that hypoxia modulates the phenotype of primary corpus cavernosum smooth muscle cells (CCSMCs) in rats, but the underlying molecular mechanism is still unknown. Platelet-derived growth factor receptor (PDGFR)-related signaling pathways are correlated with cell phenotypic transition, but research has been focused more on vascular smooth muscle and tracheal smooth muscle and less on CCSMCs. Here, we investigated the role of PDGFR-related signaling pathways in penile CCSMCs, which were successfully isolated from rats and cultured in vitro. PDGF-BB at 5, 10, or 20 ng/ml altered CCSMC morphology from the original elongated, spindle shape to a broader shape and promoted the synthetic phenotype and expression of the related proteins vimentin and collagen-I, while inhibiting the contractile phenotype and expression of the related proteins smooth muscle (SM) ?-actin (?-SMA) and desmin. Inhibition of PDGFR activity via siRNA or the PDGFR inhibitor crenolanib inhibited vimentin and collagen-I expression, increased ?-SMA and desmin expression, and considerably inhibited serine-threonine protein kinase (AKT) and signal transducer and activator of transcription 3 (STAT3) phosphorylation. STAT3 knockdown promoted the contractile phenotype, inhibited vimentin and collagen-I expression, and increased ?-SMA and desmin expression, whereas AKT knockdown did not affect phenotype-associated proteins. STAT3 overexpression in CCSMC cells weakened the suppressive effect of PDGFR inhibition on the morphology and phenotypic transformation induced by PDGF-BB. Through activation of the PDGFR/STAT3 signaling pathway, PDGF promoted the synthetic phenotype transition; thus, regulation of this pathway might contribute to ED therapy.

SUBMITTER: Yan JF

PROVIDER: S-EPMC5330473 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The platelet-derived growth factor receptor/STAT3 signaling pathway regulates the phenotypic transition of corpus cavernosum smooth muscle in rats.

Yan Jun-Feng JF Huang Wen-Jie WJ Zhao Jian-Feng JF Fu Hui-Ying HY Zhang Gao-Yue GY Huang Xiao-Jun XJ Lv Bo-Dong BD

PloS one 20170228 2

Erectile dysfunction (ED) is a common clinical disease that is difficult to treat. We previously found that hypoxia modulates the phenotype of primary corpus cavernosum smooth muscle cells (CCSMCs) in rats, but the underlying molecular mechanism is still unknown. Platelet-derived growth factor receptor (PDGFR)-related signaling pathways are correlated with cell phenotypic transition, but research has been focused more on vascular smooth muscle and tracheal smooth muscle and less on CCSMCs. Here, ...[more]

PMID: 28245285

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Project description:ObjectiveTo construct a corpus cavemosum smooth muscle cell (CCSMCs) line with TEAD1 knockout from diabetic rats with erectile dysfunction (ED) using CRISPR/Cas9 technology and explore the role of TEAD1 in phenotypic modulation of CCSMCs in diabetic rats with ED.ObjectiveModels of diabetic ED were established in male Sprague-Dawley rats by intraperitoneal injection of streptozotocin. CCSMCs from the rat models were primarily cultured and identified with immunofluorescence assay. Three sgRNAs (sgRNA-1, sgRNA-2 and sgRNA-3) were transfected via lentiviral vectors into 293T cells to prepare the sgRNA-Cas9 lentivirus. CCSMCs from diabetic rats with ED were infected by the lentivirus, and the cellular expression of TEAD1 protein was detected using Western blotting. In CCSMCs infected with the sgRNA-Cas9 lentivirus (CCSMCs-sgRNA-2), or the empty lentiviral vector (CCSMCs-sgRNA-NC) and the blank control cells (CCSMCs-CK), the expressions of cellular phenotypic markers SMMHC, calponin and PCNA at the mRNA and protein levels were detected using real-time fluorescence quantitative RT-PCR (qRT-PCR) and Western blotting, respectively.ObjectiveThe primarily cultured CCSMCs from diabetic rats with ED showed a high α-SMA-positive rate of over 95%. The recombinant lentivirus of TEAD1-sgRNA was successfully packaged, and stable TEAD1-deficient CCSMC lines derived from diabetic rat with ED were obtained. Western blotting confirmed that the protein expression of TEAD1 in TEAD1-sgRNA-2 group was the lowest (P < 0.05), and this cell line was used in subsequent experiment. The results of qRT-PCR and Western blotting showed significantly up-regulated expressions of SMMHC and calponin (all P < 0.05) and down-regulated expression of PCNA (all P < 0.05) at both the mRNA and protein levels in TEAD1-deficient CCSMCs from diabetic rats with ED.ObjectiveWe successfully constructed a stable CCSMCs line with CRISPR/Cas9-mediated TEAD1 knockout from diabetic rats with ED. TEAD1 gene knockout can induce phenotype transformation of the CCSMCs from diabetic rats with ED from the synthetic to the contractile type.

Project description:IntroductionCavernosal nerve (CN) injury is commonly caused by radical prostatectomy surgery, and it might directly lead to erectile dysfunction (ED). Currently, the role of mitogen-activated protein kinase (MAPK) family proteins in phenotypic transformation of corpus cavernosum smooth muscle cell (CCSMC) after CNs injury is poorly understood.AimTo investigate the role of p38 MAPK in hypoxia-induced phenotypic transformation of CCSMCs after CN injury.MethodsIn total, 20 Sprague-Dawley rats (male and 8 weeks of age) were randomly divided into 2 groups, including a sham group and CNCI group. In the sham group, rats were sham-operated by identifying 2 CNs without causing direct damage to the CNs. In the CNCI group, rats were subjected to bilateral CN crush injury. CCSMCs were isolated from the normal corpus cavernosum tissues of the Sprague-Dawley rat and then cultured in 21% or 1% O2 concentration context for 48 hours.Main outcome measuresIntracavernous pressure/mean arterial pressure were analyzed to measure erectile response. The impact of hypoxia on penile pathology, as well as the expression of extracellular signal-regulated kinases, the c-Jun NH2-terminal kinase, and p38 MAPK, were analyzed.ResultsCompared with the sham group, the intracavernous pressure/mean arterial pressure rate and α-smooth muscle actin expression of CNCI group were decreased significantly (P = .0001; P = .016, respectively), but vimentin expression was significantly increased (P = .023). Phosphorylated p38 level in CNCI group was decreased significantly (P = .017; sham: 0.17 ± 0.005; CNCI: 0.14 ± 0.02). The CCSMCs in the normoxia group were long fusiform, whereas the morphology of CCSMCs in the hypoxia group became hypertrophic. After hypoxia for 48 hours, the expression of α-smooth muscle actin and phosphorylated p38 MAPK was decreased significantly (P = .01; P = .024, normoxia: 0.66 ± 0.18, hypoxia: 0.26 ± 0.08, respectively), and the expression of hypoxia-inducible factor-1α and collagen I was increased significantly in hypoxia group (P = .04; P = .012, respectively).ConclusionsHypoxia induced the phenotypic transformation of CCSMCs after CNCI might be associated with the downregulation of phosphorylated p38 MAPK. Chen S, Huang X, Kong X, et al. Hypoxia-Induced Phenotypic Transformation of Corpus Cavernosum Smooth Muscle Cells After Cavernous Nerve Crush Injury by Down-Regulating p38 Mitogen-Activated Protein Kinase Expression. Sex Med 2019;7:433-440.

Project description:Platelet-derived growth factor-BB activates platelet-derived growth factor receptor-? and promotes vascular smooth muscle cell phenotypic transformation. Elevated levels of non-muscle myosin IIB (SMemb) are found in secretory smooth muscle cells along with inflammatory mediators, such as intercellular adhesion molecule-1, which can amplify neutrophil infiltration into the brain. In the present study, we investigated the role of platelet-derived growth factor-BB/platelet-derived growth factor receptor-? following intracerebral hemorrhage-induced brain injury in mice, with emphasis on its ability to promote vascular smooth muscle cell phenotypic transformation followed by increased intercellular adhesion molecule-1 expression and elevated neutrophil infiltration in the vicinity of the hematoma. We also determined the extent to which plasmin from the hematoma influences the platelet-derived growth factor-BB/platelet-derived growth factor receptor-? system subsequent to intracerebral hemorrhage.Controlled in vivo laboratory study.Animal research laboratory.One hundred and fifty six eight-week-old male CD1 mice.Brain injury was induced by autologous arterial blood or plasmin injection into mouse brains. Small interfering RNA targeting platelet-derived growth factor receptor-? was administered 24 hours before intracerebral hemorrhage. A platelet-derived growth factor receptor antagonist, Gleevec, was administered following intracerebral hemorrhage. A mitogen-activated protein kinase-activated protein kinase 2 inhibitor (KKKALNRQLGVAA) was delivered with platelet-derived growth factor-BB in naïve animals. Platelet-derived growth factor-BB was injected with a plasmin inhibitor (?-aminocaproic acid) in intracerebral hemorrhage mice. Plasmin-injected mice were given platelet-derived growth factor receptor-? small interfering RNA 24 hours before the operation. Neurological deficits, brain edema, western blots, and immunofluorescence were evaluated.Platelet-derived growth factor receptor-? small interfering RNA attenuated SMemb and intercellular adhesion molecule-1 expression and neutrophil infiltration at 24 hours post injury and reduced neurological deficits and brain edema at 24 and 72 hours following intracerebral hemorrhage. The platelet-derived growth factor receptor antagonist, Gleevec, reduced SMemb and intercellular adhesion molecule-1 expression. Platelet-derived growth factor receptor-? activation led to increased expression of intercellular adhesion molecule-1 and was reversed by KKKALNRQLGVAA in naïve mice. Plasmin inhibition suppressed platelet-derived growth factor receptor-? activation and neutrophil infiltration, whereas exogenous platelet-derived growth factor-BB increased platelet-derived growth factor receptor-? activation, regardless of plasmin inhibition. Platelet-derived growth factor receptor-? small interfering RNA decreased the expression of intercellular adhesion molecule-1 by plasmin injection.The platelet-derived growth factor-BB/platelet-derived growth factor receptor-? system contributes to neuroinflammation through vascular smooth muscle cell phenotypic transformation near the hematoma via the p38 mitogen-activated protein kinase/mitogen-activated protein kinase-activated protein kinase 2 pathway following intracerebral hemorrhage. Plasmin is hypothesized to be upstream of the proposed neuroinflammatory system. The therapeutic intervention targeting the platelet-derived growth factor-BB/platelet-derived growth factor receptor-? is a novel strategy to prevent plasmin-induced brain injury following intracerebral hemorrhage.

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The platelet-derived growth factor receptor/STAT3 signaling pathway regulates the phenotypic transition of corpus cavernosum smooth muscle in rats.

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The platelet-derived growth factor receptor/STAT3 signaling pathway regulates the phenotypic transition of corpus cavernosum smooth muscle in rats.

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