Microfibrillar-associated protein 5 suppresses adipogenesis by inhibiting essential coactivator of PPARγ

Abstract

Femoral head necrosis is responsible for severe pain and its incidence is increasing. Abnormal adipogenic differentiation and fat cell hypertrophy of bone marrow mesenchymal stem cells increase intramedullary cavity pressure, leading to osteonecrosis. By analyzing gene expression before and after adipogenic differentiation, we found that Microfibril-Associated Protein 5 (MFAP5) is significantly down-regulated in adipogenesis whilst the mechanism of MFAP5 in regulating the differentiation of bone marrow mesenchymal stem cells is unknown. The purpose of this study was to clarify the role of MAFP5 in adipogenesis and therefore provide a theoretical basis for future therapeutic options of osteonecrosis. By knockdown or overexpression of MFAP5 in C3H10 and 3T3-L1 cells, we found that MFAP5 was significantly down-regulated as a key regulator of adipogenic differentiation, and identified the underlying downstream molecular mechanism. MFAP5 directly bound to and inhibited the expression of Staphylococcal Nuclease And Tudor Domain Containing 1, an essential coactivator of PPARγ, exerting an important regulatory role in adipogenesis.

Figure 1

Association of MFAP5 with adipogenic differentiation. (A) Fifty-seven genes were at the intersection of three sequencing datasets before and after adipogenic differentiation. (B) Radar chart of 25 genes with the highest differential expression. Blue and red, down- and up-regulated during adipogenesis, respectively. Multiples of gene expression in each database are indicated by different colors. (C) Oil red O staining of C3H10 and 3T3-L1 cells. (D) Endogenous expression and expression pattern during adipogenesis of MFAP5 in C3H10 and 3T3-L1 cells. (E–G) Relative mRNA levels of MFAP5, CEBPα, FABP4, and SREB1 during adipogenesis. n = 3, the experiment was repeated 3 times. Values are means ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2

Establishment of MFAP5-knockdown C3H10 and 3T3-L1 cells and the role of MFAP5 in adipogenesis. (A) Western blotting of MFAP5 protein levels in the blank, control, and MFAP5-shRNA1-3 groups of C3H10 and 3T3-L1 cells. (B–C) Quantification of protein levels using ImageJ software in the blank, control, and MFAP5-shRNA1-3 groups of C3H10 and 3T3-L1 cells, respectively. (D–E) qRT-PCR of MFAP5 mRNA levels in the blank, control, and MFAP5 -shRNA1-3 groups of C3H10 and 3T3-L1 cells. (F) Lipid droplets accumulation determined by Oil red O staining. (G) Absorbance at 510 nm of Oil red O-stained cells. (H–J) Expression of CEBPα, FABP4, and SREB1 during adipogenesis was suppressed in MFAP5-knockdown cells. n = 3, the experiment was repeated 3 times. Values are means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

MFAP5 directly bound to and suppressed SND1, inhibiting activation of the PPARγ signaling pathway. (A and B) Co-immunoprecipitation showed MFAP5 and SND1 interacted at the protein level with an IgG antibody as a negative control in C3H10 and 3T3-L1 cells. (C) Knockdown of MFAP5 up-regulated SND1 expression. (D) During adipogenesis, SND1 expression was up-regulated and bound to PPARγ in C3H10 and 3T3-L1 cells. (E) CD36 and Adipsin (downstream genes of PPARγ) were significantly up-regulated in MFAP5-knockdown cells. n = 3, the experiment was repeated 3 times.

Figure 4

Silencing of SND1 in MFAP5-knockdown cell lines reversed the promotion of adipogenesis. (A) Western blotting of SND1 in MFAP5-knockdown cells compared to the control. (B) SND1 knockdown reversed the activation of PPARγ downstream proteins, including CD36 and Adipsin, in MFAP5-knockdown cells before and after adipogenic induction. (C) Accumulation of lipid droplets by Oil red O staining 10 days after adipogenic induction in the control, MFAP5-sh, and MFAP5-SND1-sh groups. (D–F) Relative expression levels of adipogenic biomarkers—CEBPα, FABP4, and SREB1—by qRT-PCR on day 10 of adipogenesis. n = 3, the experiment was repeated 3 times. Values are means ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5

MFAP5 overexpression suppressed adipogenesis by inhibiting SND1. (A–C) Establishment of MFAP5-overexpressing C3H10 and 3T3-L1 cells. (D) Lipid droplets accumulation was significantly reduced in the MFAP5-overexpressing group. (E) Expression of SND1, CD36, and Adipsin during adipogenic induction in the control and MFAP5-overexpressing groups. (F–H). Relative mRNA levels of CEBPα, FABP4 and SREB1 showed that MFAP5 overexpression hindered adipogenic differentiation of C3H10 and 3T3-L1 cells. I. Absorbance at 510 nm of Oil red O-stained cells. n = 3, the experiment was repeated 3 times. Values are means ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.