miR-135b expression downregulates Ppm1e to activate AMPK signaling and protect osteoblastic cells from dexamethasone

Abstract

Activation of AMP-activated protein kinase (AMPK) could potently protect osteoblasts/osteoblastic cells from dexamethasone (Dex). We aim to induce AMPK activation via microRNA ("miRNA") downregulation of its phosphatase Ppm1e. We discovered that microRNA-135b ("miR-135b") targets the 3' untranslated regions (UTRs) of Ppm1e. In human osteoblasticOB-6 cells and hFOB1.19 cells, forced-expression of miR-135b downregulated Ppm1e and activated AMPK signaling. miR-135b also protected osteoblastic cells from Dex. shRNA-induced knockdown of Ppm1e similarly activated AMPK and inhibited Dex-induced damages. Intriguingly, in the Ppm1e-silenced osteoblastic cells, miR-135b expression failed to offer further cytoprotection against Dex. Notably, AMPK knockdown (via shRNA) or dominant negative mutation abolished miR-135b-induced AMPK activation and cytoprotection against Dex. Molecularly, miR-135b, via activating AMPK, increased nicotinamide adenine dinucleotide phosphate (NADPH) activity and inhibited Dex-induced oxidative stress. At last, we found that miR-135b level was increased in human necrotic femoral head tissues, which was correlated with Ppm1e downregulation and AMPK activation. There results suggest that miR-135b expression downregulates Ppm1e to activate AMPK signaling, which protects osteoblastic cells from Dex.

Keywords: AMP-activated protein kinase (AMPK); dexamethasone (Dex); microRNA-135b; osteoblastic cells; phosphatase 1E (Ppm1e).

Figure 1. Forced expression of microRNA-135b downregulates Ppm1e but activates AMPK signaling in human osteoblastic cells

A. microRNA-135b (“miR-135b”) targets the 3' untranslated regions (UTRs, position 517-524) of human Ppm1e (Figure 1A). Human osteoblastic OB-6 cells B-D. or hFOB1.19 cells E-G. were transfected with microRNA-135b (“miR-135b”) or non-sense control microRNA (“miR-C”), and stable cells were established. Expressions of miR-135b (B and E) and Ppm1e mRNA (C and F) were tested by quantitative real-time PCR (“qRT-PCR”) assay; Expression of listed proteins in these cells were tested by Western blot assay (D and G). Experiments in this figure were repeated four times, and similar results were obtained. “Trans” stands for transfection reagents only (B-G). *p<0.05 vs. group “miR-C” (B, C, E and F).

Figure 2. Forced expression of microRNA-135b protects osteoblastic cells from Dex

Stable osteoblastic OB-6 cells A-C. or hFOB1.19 cells D-F. expressing microRNA-135b (“miR-135b”) or non-sense control microRNA (“miR-C”) were treated with or without Dex (1 μM) for 24 hours, cell viability (MTT assay, A and D), apoptosis (Histone DNA ELISA assay, B and E) and cell death (trypan blue assay, C and F) were tested. Experiments in this figure were repeated four times, and similar results were obtained. “Ctrl” stands for untreated control group. “Trans” stands for transfection reagents only. *p<0.05 vs. “miR-C” cells with Dex treatment.

Figure 3. shRNA knockdown of Ppm1e activates AMPK and protects osteoblastic cells from Dex

OB-6 cells were infected with lentiviral Ppm1e shRNA (“−1 or −2”) or non-sense control shRNA (“scr shRNA”), and stable cells were established. miR-135b and Ppm1e mRNA levels were tested (A, qRT-PCR assay), Ppm1e protein expression and AMPK activation (p-AMPK/p-ACC) were also tested (B, Western blot assay). Above cells were treated with or without Dex (1 μM) for 24 hours, cell viability (C, MTT assay) and apoptosis (D, Histone DNA ELISA assay) were shown. Ppm1e shRNA (“-1”)-expressing OB-6 cells were transfected with miR-135b expressing construct, expressions of miR-135b E. and Ppm1e mRNA F. were tested by qRT-PCR assay, these cells were also treated with or without Dex (1 μM) for 24 hours, cell viability G. and apoptosis H. were shown. “Ctrl” stands for untreated control group. Experiments in this figure were repeated four times, and similar results were obtained. *p<0.05 vs. “scr shRNA” cells (A, C-H).

Figure 4. AMPK knockdown or mutation abolishes miR-135b-induced cytoprotection in osteoblastic cells

miR-135b expressing OB-6/hFOB1.19 cells cells were constructed with AMPKα shRNA, dominant negative AMPKα (“dn-AMPKα-flag”, T172A) or the scramble control shRNA (“Scr shRNA”), expressions of listed proteins in these cells were tested by Western blots (A, for OB-6 cells); miR-135b and Ppm1e mRNA expressions were also tested (B, qRT-PCR assay, for OB-6 cells). Above cells were treated with or without Dex (1 μM) for indicated periods of time, cell viability (MTT assay, C and E), apoptosis intensity (Histone DNA ELISA assay, D and F) and ROS content (DCFH-DA fluorescent dye assay, G) were tested. NADPH activity in above cells was also shown H. Experiments in this figure were repeated four times, and similar results were obtained. “Ctrl” stands for untreated control group. ^#p<0.05.

Figure 5. Upregulation of miR-135b in patients' osteonecrosis tissues

Western blots analyzing the expression of listed proteins in surgery isolated femoral head tissues (both normal and necrotic) from GC-taking patients; ACC phosphorylation A. and Ppm1e protein (D, normalized to Tubulin) were quantified. Expressions of miR-135b B. and Ppm1e mRNA C. were also shown (qRT-PCR assay) in above tissues. *p<0.05 vs. “S” tissues.