Cldn4overexpression promotes penile cavernous smooth muscle cell fibrotic response via the JNK signaling pathway

AbstractBackgroundErectile dysfunction (ED), defined as the inability to achieve or maintain a penile erection sufficient to satisfy sexual behavior, is prevalent worldwide.AimUsing previous research, bioinformatics, and experimental confirmation, we aimed to discover genes that contribute to ED through regulating hypoxia in corpus cavernosum smooth muscle cells (CCSMCs).MethodsWe used the Gene Expression Omnibus to acquire the sequencing data of the corpus cavernosum transcriptome for diabetic ED and nerve injury type ED rats. We intersected the common differentially expressed genes. Further verification was performed using single cell sequencing. Real-time quantitative polymerase chain reaction and immunofluorescence were used to investigate whether the differentially expressed genes are found in the corpus cavernosum. We used induced hypoxia to assess cell viability changes, and we developed a lentivirus overexpressing Cldn4 for in vitro and in vivo experiments to measure changes in JNK signaling, fibrosis, hypoxia, and erectile function.OutcomesOur results indicate that targeting the JNK pathway and decreasing local hypoxia may be better options for therapeutic intervention to improve erectile function.ResultsWe identified Cldn4 and found its expression increased in the corpora cavernosa of the 2 datasets. In addition, we found that hypoxia can increase the expression of Cldn4, activate the JNK signaling pathway, and exacerbate fibrosis in CCSMCs. Cldn4 overexpression in CCSMCs activated the JNK signaling pathway and increased fibrotic protein expression. Last, rat corpus cavernosum overexpressing Cldn4 activated the JNK signaling pathway, increased local fibrosis, and impaired erectile function.Clinical ImplicationsThrough bioinformatics and in vitro and in vivo experiments, we found that Cldn4 has a negative effect on ED, and targeting Cldn4 may provide new ideas for ED treatment.Strengths and LimitationsAlthough we have identified Cldn4 as a potential target for ED treatment, we have only conducted preliminary validation on CCMSCs, and we still need to further validate in other cell lines.ConclusionCCSMC hypoxia leads to increased Cldn4, in both nerve injury and diabetic ED rat models, and promotes fibrosis by activating the JNK signaling pathway.