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. Author manuscript; available in PMC: 2017 Apr 4.
Published in final edited form as: J Bone Miner Res. 2015 Jun;30(6):1044–1052. doi: 10.1002/jbmr.2438

Fig. 5.

Fig. 5

miR-140 regulate MEF2C expression via the p38 MAPK pathway. (A) Mir140-null chondrocytes show increased Dnpep, RalA, Creb3l1, and Rac1 transcripts. qRT-PCR was performed on primary rib chondrocytes form Mir140+/− and Mir140−/− mice. (B) RalA protein expression is increased in Mir140-null chondrocytes, whereas Sox9 expression is not altered. (C) Overexpression of Dnpep, human RalA (hRalA), Creb3l1, or a constitutively active mutant of Rac1 (caRac1) does not increase Mef2c expression. Gene expression was determined byqRT-PCR 3days after retrovirus infection to overexpress indicated genes. Infected viruses are indicated below the x-axis. Retrovirus-mediated gene transfer achieved twofold to threefold upregulation of indicated genes. Human RalA transcript was not detected in control (GFP) (ND). (D) p38 MAPK signaling is upregulated in Mir140-null chondrocytes. Phosphorylation of p38 MAPK (p-p38 MAPK) and ERK1/2 (p-ERK) was assessed by immunoblot analysis at indicated time points after serum stimulation. A representative result from three independent experiments using pairs of control and Mir140-null littermates is shown. (E) Quantification of the p-p38 level at the 5-min time point in D. Relative p-p38 levels in Mir140−/− (−/−) and Mir140+/− (+/−) were calculated in three sets of independent experiments. *p < 0.05, n = 3. (F) Retrovirus-mediated miR-140 overexpression reduces basal and stimulated levels of p-p38 MAPK. Mir140-null primary rib chondrocytes were infected with control (GFP) and miR-140-GFP (140) retroviruses and cultured for 2 days. p-p38 levels were determined in a similar fashion as in D. Cell lysates were also prepared from cells cultured in a growth medium without serum starvation (Growth). Signals were quantified, normalized, and expressed relative to control. (G) p38 MAPK inhibition reduces expression of MEF2C in a dose-dependent manner, whereas expression of HDAC4 and Histone 2 A (H2A) is not affected. Cells were cultured for 48 hours in the presence of indicated concentration of SB 203580. Signals were quantified, normalized, and expressed relative to vehicle control. (H) Proposed model. miR-140 enhances the PTHrP/HDAC4 pathway to control hypertrophic differentiation by suppressing premature MEF2C expression, at least in part, through dampening p38 MAPK signaling. ND = not detected.