Neuropilin 1 (NRP1) Positively Regulates Adipogenic Differentiation in C3H10T1/2 Cells

Abstract

Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for several ligands, is highly expressed in many kinds of mesenchymal stem cells (MSCs), but its function is poorly understood. In this study, we explored the roles of full-length NRP1 and glycosaminoglycan (GAG)-modifiable NRP1 in adipogenesis in C3H10T1/2 cells. The expression of full-length NRP1 and GAG-modifiable NRP1 increased during adipogenic differentiation in C3H10T1/2 cells. NRP1 knockdown repressed adipogenesis while decreasing the levels of Akt and ERK1/2 phosphorylation. Moreover, the scaffold protein JIP4 was involved in adipogenesis in C3H10T1/2 cells by interacting with NRP1. Furthermore, overexpression of non-GAG-modifiable NRP1 mutant (S612A) greatly promoted adipogenic differentiation, accompanied by upregulation of the phosphorylated Akt and ERK1/2. Taken together, these results indicate that NRP1 is a key regulator that promotes adipogenesis in C3H10T1/2 cells by interacting with JIP4 and activating the Akt and ERK1/2 pathway. Non-GAG-modifiable NRP1 mutant (S612A) accelerates the process of adipogenic differentiation, suggesting that GAG glycosylation is a negative post-translational modification of NRP1 in adipogenic differentiation.

Keywords: adipogenic differentiation; mesenchymal stem cells; neuropilin 1.

Figure 1

Expression and subcellular localization of NRP1 during adipogenesis in C3H10T1/2 cells. (A) C3H10T1/2 cells have the capacity to differentiate into adipocytes. (B) C3H10T1/2 cells were cultured in an adipogenic medium for the indicated days; Western blot analysis was performed using anti-NRP1 antibody. (C) C3H10T1/2 cells were cultured in an adipogenic medium for three days; immunofluorescence staining and confocal laser scanning microscopy detected NRP1, β-actin, and DAPI. Scale bars, 20 µm. The images are representative of at least three independent experiments.

Figure 2

Effect of NRP1 knockdown on adipogenesis in C3H10T1/2 cells. (A,B) qPCR and Western blot analysis for NRP1 were performed in NRP1-knockdown cells. (C) Oil red O staining of NRP1-knockdown cells with adipogenic induction for four days. Scale bars, 200 µm. (D) Lipid accumulation was assessed by quantification of oil red O staining. (E) qPCR analysis for Cebpa, Pparg, Fabp4, and Adipoq in sh-cont and sh-2-1cells with or without adipogenic induction for four days. (F) NRP1-knockdown cells were cultured in an adipogenic medium for the indicated time, and Western blot analysis was performed using anti-Akt, phosphorylated Akt, ERK and phosphorylated ERK antibodies. The images are representative of at least three independent experiments. Control medium (CM); differentiation medium (DM). The data are presented as mean ± standard deviation (SD) (n = 3 independent experiments) * p < 0.05.

Figure 3

Effect of JIP4 silencing on adipogenesis in C3H10T1/2 cells. (A) C3H10T1/2 cells were cultured in an adipogenic medium for the indicated days, and Western blot analysis was performed using anti-JIP4 antibody. (B,C) qPCR and Western blot analysis for JIP4 were performed in NRP1-JIP4–double-knockdown cells. (D) Oil red O staining of NRP1-JIP4–double-knockdown cells with adipogenic induction for four days. Scale bars, 200 µm. (E) Lipid accumulation was assessed by quantification of oil red O staining. (F) qPCR analysis for Cebpa, Pparg, Fabp4, and Adipoq in NRP1-JIP4–double-knockdown cells with or without adipogenic induction for four days. (G) JIP4-knockdown cells were cultured in an adipogenic medium for the indicated time, and Western blot analysis was performed using anti–Akt, phosphorylated Akt, ERK and phosphorylated ERK antibodies. The images are representative of at least three independent experiments. The data are presented as mean ± standard deviation (SD) (n = 3 independent experiments) * p < 0.05.

Figure 4

NRP1 binds to JIP4 in adipogenic differentiation. (A,B) qPCR and Western blot analysis for NRP1 were performed in NRP1 wild-type (WT) flag-overexpressing cells and control cells. Samples from ov-NRP1 WT and ov-cont cells from C3H10T1/2 cells (C) or HEK 293T cells (D) were immunoprecipitated with anti-Flag antibody–conjugated agarose and subjected to Western blot analysis. Whole-cell lysate from the cells was used as a positive control (input). The blotted membranes were incubated with anti-NRP1 antibody (upper panel) or anti-JIP4 antibody (lower panel). The images are representative of at least three independent experiments. Immunoprecipitation (IP); control medium (CM); differentiation medium (DM). The data are presented as mean ± standard deviation (SD) (n = 3 independent experiments) * p < 0.05.

Figure 5

Effects of nonmodifiable NRP1 mutant (S612A) on adipogenic differentiation. (A) Design of lentivirus constructs. Ser612 exists in the bridge region between the b1b2 and MAM domains. (B) Western blot analysis for NRP1 was performed in ov-NRP1 WT and ov-NRP1 S612A cells. (C) Oil red O staining of ov-cont, ov-NRP1 WT, and ov-NRP1 S612A cells with adipogenic induction for four days. Scale bars, 200 µm. (D) Lipid accumulation was assessed by quantification of oil red O staining. (E) qPCR analysis for Cebpa, Pparg, Fabp4, and Adipoq in ov-cont, ov-NRP1 WT, and ov-NRP1 S612A cells with adipogenic induction for four days. (F) Ov-cont, ov-NRP1 WT, and ov- NRP1 S612A cells were cultured in an adipogenic medium for the indicated time, and Western blot analysis was performed using anti–Akt, phosphorylated Akt, ERK, and phosphorylated ERK antibodies. The images are representative of at least three independent experiments. The data are presented as mean ± standard deviation (SD) (n = 3 independent experiments) * p < 0.05.

Figure 6

Schematic of the NRP1–JIP4–Akt/ERK signaling pathway promoting adipogenic differentiation of C3H10T1/2 cells.