A microRNA signature for a BMP2-induced osteoblast lineage commitment program - PubMed (original) (raw)

A microRNA signature for a BMP2-induced osteoblast lineage commitment program

Zhaoyong Li et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Bone morphogenetic proteins (BMPs) are potent morphogens that activate transcriptional programs for lineage determination. How BMP induction of a phenotype is coordinated with microRNAs (miRNAs) that inhibit biological pathways to control cell differentiation, remains unknown. Here, we show by profiling miRNAs during BMP2 induced osteogenesis of C2C12 mesenchymal cells, that 22 of 25 miRNAs which significantly changed in response to BMP2 are down-regulated. These miRNAs are each predicted to target components of multiple osteogenic pathways. We characterize two representative miRNAs and show that miR-133 directly targets Runx2, an early BMP response gene essential for bone formation, and miR-135 targets Smad5, a key transducer of the BMP2 osteogenic signal, controlled through their 3'UTR sequences. Both miRNAs functionally inhibit differentiation of osteoprogenitors by attenuating Runx2 and Smad5 pathways that synergistically contribute to bone formation. Although miR-133 is known to promote MEF-2-dependent myogenesis, we have identified a second complementary function to inhibit Runx2-mediated osteogenesis. Our key finding is that BMP2 controls bone cell determination by inducing miRNAs that target muscle genes but mainly by down-regulating multiple miRNAs that constitute an osteogenic program, thereby releasing from inhibition pathway components required for cell lineage commitment. Thus, our studies establish a mechanism for BMP morphogens to selectively induce a tissue-specific phenotype and suppress alternative lineages.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

A program of miRNAs is expressed during C2C12 osteogenic differentiation. (A and B) Expression profile of osteoblastic and myogenic markers during C2C12 differentiation. (A) Western blot of Runx2 and β-actin (as control) expression in control and BMP2-treated groups. Right shows Runx2 densitometry quantitation normalized to β-actin. (B) mRNA levels (Q-PCR normalized to GAPDH) of osteoblastic markers Runx2, alkaline phosphatase (AP), osteocalcin (OC), and fibromodulin (Fmod), and muscle related genes myogenin (Myog) and cadherin 15 (Cdh 15) during the time course experiments used for miRNA profiling. Relative levels are expressed setting 0 time to 1 (B). (C and D) BMP2 down-regulated (C) and up-regulated (D) miRNAs from miRNA profiling data combined from two independent experiments. For presentation of hierarchical clustered gene, dChip software was used. See Materials and Methods for normalization of each miRNA data point.

Fig. 2.

miR-133 and miR-135 expression during BMP2-induced C2C12 osteogenic differentiation. Validation of the array data shown for miR-133 (A and B) and miR-135 (C and D) was performed with RNA from replicate time course up to 48 h. Northern blot analysis of miR-133 and miR-135 expression in control or BMP2 treatment is shown (A and C). U6 snRNA was used as a loading control and for normalization after densitometric quantitation plotted in B and D.

Fig. 3.

miR-133 and miR-135 functional activity on target genes. (A) Schematic of miR-133 putative target site in mouse Runx2 3′UTR and alignment of miR-133a with wild-type (WT) and mutant (MUT) 3′UTR region of Runx2 showing complementary pairing. The 3 mutated nucleotides are underlined. (B) MC3T3 cells were cotransfected with the luciferase reporters carrying wild-type Runx2 3′UTR or mutated Runx2 3′UTR, phRL-null (Renilla plasmid) and 100 nM RNA oligonucleotides of miR-Control (miR-C), the miR-133a or miR-34c as an irrelevant control. Effects of mir-133a and control miRNAs on the reporter constructs were shown after 36 h. The ratio of reporter (Firefly) to control phRL-null plasmid (Renilla) in relative luminescence units was plotted. Error bars represent the standard error for n = 3. (C) miR-133 directly targets and regulates Runx2 and inhibits osteoblastogenesis. MC3T3 osteoblast cells were transfected with miR-133a, miRNA-Control or transfection reagent only (Mock) at the indicated concentrations. Western blots for Runx2 and Lamin B1 (as control) were performed on total cell lysates collected at 48 h. (D) Quantitative mRNA levels (normalized by GAPDH) by Q-PCR for Runx2, alkaline phosphatase (Alk Phos), osteocalcin (OC) and histone H4 in 100 nM oligo transfection. Values represent mean ± SD of n = 3 from two independent experiments. (E) Schematic of the miR-135 putative target site in the mouse Smad5 mRNA 3′UTR and alignment of miR-135a with wild-type (WT) and mutant (MUT) 3′ UTR region of Smad5. Complementary pairing between miR-135a and Smad5 is illustrated. Mutated 3′UTR nucleotides (n = 3) are underlined. (F) Functional activity of the luciferase reporter plasmid carrying wild-type or mutated Smad5 3′UTR was assessed as described above in B. Error bars represent the standard error for n = 3. (G) MC3T3 cells were transfected with miR-135a and miRNA-Control as described in C. (H) The mRNA levels of osteoblast marker genes after transfection of miR-Control and miR-135a were determined as described in D.

Fig. 4.

miR-133 and miR-135 inhibit BMP2-induced C2C12 osteogenic differentiation. (A) miR-133a overexpression restrained BMP2-induced Runx2, p-Smad1/5, and p27 protein increase. C2C12 cells transfected with 100 nM miR-133a and miRNA-Control (miR-C) for 12 h, then cultured in 0.25% albumin serum-free medium, which contains 300 ng/ml BMP2. Western blots for indicated proteins were performed on total cell lysates as shown. (B) miR-133 inhibits osteoblast markers Smad5, alkaline phosphatase (Alk Phos), Osteocalcin, and Hoxa10 mRNA (Q-PCR normalized by GAPDH), but has no effect on H4 and HPRT (hypoxanthine guanine phosphoribosyl transferase 1). (C) mir-135 down-regulates the BMP2 transducer Smad5. C2C12 cells transfected with 100 nM miR-135a were harvested and for consequences on the indicated proteins under conditions described in A. Western blots for Runx2, Smad5, _p_-Smad1/5, p27, MyoD, and β-actin (as control) were performed on total cell lysates as shown. (D) miR-135 decreases osteogenesis. mRNA levels of osteoblast markers Runx2, alkaline phosphatase (Alk Phos), Osteocalcin and Hoxa10, and H4 and HPRT were detected as described in B. (E) Model of miR-133 and miR-135-mediated regulation of osteoblast differentiation. MiR-133 and miR-135 targeted osteogenic factors Runx2 and Smad5, respectively. BMP2 treatment down-regulates expression of miR-133 and miR-135 and releases expression of Runx2 and Smad5 to promote osteoblast differentiation.

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A microRNA signature for a BMP2-induced osteoblast lineage commitment program - PubMed (original) (raw)