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. 2023 Feb 27;28(1):16.
doi: 10.1186/s11658-023-00433-0.

The RXFP2-PLC/PKC signaling pathway mediates INSL3-induced regulation of the proliferation, migration and apoptosis of mouse gubernacular cells

Shouxing Duan  1   2 , Xuewu Jiang  3 , Jianhong Li  4 , Maxian Fu  1 , Zhuo Li  1 , Yiyi Cheng  2 , Yangmu Zhuang  2 , Ming Yang  2 , Wenfeng Xiao  4 , Hongyan Ping  3 , Yao Xie  5 , Xiaojun Xie  6 , Xuan Zhang  7
Affiliations

The RXFP2-PLC/PKC signaling pathway mediates INSL3-induced regulation of the proliferation, migration and apoptosis of mouse gubernacular cells

Shouxing Duan et al. Cell Mol Biol Lett. .

Abstract

Background: Testicular hypoplasia can affect the sexual and reproductive ability in adulthood, and even increase the risk of cancer. Abnormal development of the gubernaculum is one of the important factors of testicular hypoplasia. Therefore, a study of the structure and function of the gubernaculum is an important but neglected new breakthrough point for investigating the normal/abnormal development of the testis. Previous findings showed that Insulin like factor 3 (INSL3) is a key factor regulating the growth of gubernaculum, however, the mechanism by which INSL3 acts on the gubernaculum remains unknown. Therefore, we probed the mechanism associated with INSL3-induced the proliferation, migration, and apoptosis of gubernacular cells in mice.

Methods: A culture cell model of neonatal mice gubernaculum is established by INSL3 intervention. We blocked PLC/PKC signaling pathway with U73122 pretreat to investigate the role of the PLC/PKC signaling pathway. The changes of cell proliferation, migration, and apoptosis were detected by molecular biological methods. In addition, the levels of PCNA and F-action were detected by immunofluorescence and western blotting.

Results: We found that INSL3 can promote the proliferation and migration of gubernacular cells and inhibit their apoptosis, meanwhile, INSL3 significantly up-regulated PLC/PKC protein phosphorylation. However, treatment with the PLC/PKC signaling pathway inhibitor U73122 significantly inhibited these effects of INSL3. Besides, we found that INSL3 could up-regulate the protein expression level of PCNA and F-actin, while the PCNA and F-actin expression was significantly weakened after U73122 pretreatment.

Conclusions: This research revealed that INSL3 binding to RXFP2 may up-regulate the expression levels of PCNA and F-actin by activating the PLC/PKC signaling pathway to promote the proliferation and migration of gubernacular cells. It suggests that the RXFP2-PLC/PKC axis may serve as a novel molecular mechanism by which INSL3 regulates growth of the gubernaculum.

Keywords: Gubernaculum; INSL3; PLC/PKC; RXFP2; Signaling pathway.

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Conflict of interest statement

The authors have declared no conflict of interests.

Figures

Fig. 1
Fig. 1
U73122 inhibited the effect of INSL3 on the proliferation of gubernacular cells. A Cell morphology was observed by HE staining. Scale bars, 50 μm. B Cell proliferation was detected by CCK8. C Cell counting measures changes in the number of cells. The counting method is shown in Additional file 2: Fig. S2. D The expression of proliferating cell nuclear antigen (PCNA) was observed by immunofluorescence. Scale bars, 20 μm. E The expression of PCNA protein was analyzed by Western blot. Data are mean ± standard deviation of three independent experiments. *&#P < 0.05, **&##P < 0.01, and ***&###P < 0.001
Fig. 2
Fig. 2
U73122 mediated the effect of INSL3 on the migration ability of gubernacular cells. A, B The scratch and transwell tests assessed gubernacular cell migration in each group. C, D The expression of fibrous actin (F-actin) protein was analyzed by immunofluorescence staining and Western blot. Scale bars, 20 μm. Data are mean ± standard deviation of three independent experiments. *&#P < 0.05, **&##P < 0.01, ***&###P < 0.001, and ****&####P < 0.0001
Fig. 3
Fig. 3
U73122 inhibited the anti-apoptotic effect of INSL3 on gubernacular cells. A, B Flow cytometry evaluate the effect of INSL3 on apoptosis of gubernacular cells. C, D Cell apoptosis was detected by TUNEL staining. Scale bars, 50 μm Data are mean ± standard deviation of three independent experiments. *&#P < 0.05, **&##P < 0.01, and ***&###P < 0.001
Fig. 4
Fig. 4
PLC/PKC was involved in the effect of INSL3 on gubernacular cells. A The change of P-PLC/PLC expression level was detected by western blotting. B The change of P-PKC/PKC expression level was detected by western blotting. Data are mean ± standard deviation of three independent experiments. *&#P < 0.05, and ***&###P < 0.001
Fig. 5
Fig. 5
Graphic abstract. INSL3 regulated gubernacular cell proliferation, migration, and apoptosis via the PLC/PKC signaling pathway. Specifically, INSL3 binding to RXFP2 may up-regulate the expression levels of PCNA and F-actin by activating the PLC/PKC signaling pathway to promote the proliferation and migration of gubernacular cells and inhibit their apoptosis
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