Identification of novel microRNA inhibiting actin cytoskeletal rearrangement thereby suppressing osteoblast differentiation
- 234 Downloads
We report the role of miR-1187 in regulation of osteoblast functions. Over-expression of miR-1187 inhibited osteoblast differentiation. Target prediction analysis tools and experimental validation by luciferase 3′ UTR reporter assay identified BMPR-II and ArhGEF-9 as direct targets of miR-1187. ArhGEF-9 activates Cdc42 which has a major role in actin reorganization. BMP-2 also induces actin polymerization. Role of miR-1187 in actin reorganization was determined by western blotting, immunofluorescence, and in vivo gene silencing studies. Reduced protein levels of BMPR-II, activated Cdc42, and downstream signaling molecules were observed in miR-1187-transfected osteoblasts. miR-1187 over-expression resulted in decreased actin polymerization. Additionally, P-cofilin, which does not bind F-actin, was decreased in miR-1187-transfected cells. These results were corroborated by administration of BMPR-II exogenously in miR-1187-transfected osteoblasts. Silencing of miR-1187 in neonatal mice mitigated all the inhibitory effects of miR-1187 on actin cytoskeletal rearrangement. Importantly, in vivo treatment of miR-1187 inhibitor to ovariectomized BALB/c mice led to significant improvement in trabecular bone microarchitecture. Overall, miR-1187 functions as a negative regulator of osteogenesis by repressing BMPR-II and ArhGEF-9 expression thus suppressing non-Smad BMP2/Cdc42 signaling pathway and inhibiting actin reorganization. miR-1187 functions as a negative regulator of osteogenesis by repressing BMPR-II expression, which in turn, suppresses non-Smad BMP2/Cdc42 signaling pathway, thus inhibiting actin cytoskeletal rearrangement. Silencing of miR-1187 significantly improves trabecular bone microarchitecture. As miR-1187 exerts a negative regulatory role in osteoblasts function, hence, we propose that therapeutic approaches targeting miR-1187 could be useful in enhancing the bone formation and treatment of pathological conditions of bone loss.
KeywordsMicroRNA BMPR-II BMP signaling Osteoblast differentiation Actin Cell migration
Experiments were conceived and designed by DS and AAJ. AAJ performed the experiments and analyzed the data. DS wrote the paper. RP and JK assisted in the experiments and helped in the analysis of the data. All authors have read and approved the manuscript before submission. The CDRI communication number is 9641.
Fellowship grants from the Council of Scientific and Industrial Research (AAJ and JK), University Grants Commission (RP), Government of India are acknowledged.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 3.Luu HH, Song WX, Luo X, Manning D, Luo J, Deng ZL, Sharff KA, Montag AG, Haydon RC, He TC (2007) Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society 25:665–677CrossRefGoogle Scholar
- 7.Hassan MQ, Maeda Y, Taipaleenmaki H, Zhang W, Jafferji M, Gordon JA, Li Z, Croce CM, van Wijnen AJ, Stein JL et al (2012) miR-218 directs a Wnt signaling circuit to promote differentiation of osteoblasts and osteomimicry of metastatic cancer cells. J Biol Chem 287:42084–42092CrossRefPubMedPubMedCentralGoogle Scholar
- 8.Inose H, Ochi H, Kimura A, Fujita K, Xu R, Sato S, Iwasaki M, Sunamura S, Takeuchi Y, Fukumoto S, Saito K, Nakamura T, Siomi H, Ito H, Arai Y, Shinomiya KI, Takeda S (2009) A microRNA regulatory mechanism of osteoblast differentiation. Proc Natl Acad Sci U S A 106:20794–20799CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Kureel J, Dixit M, Tyagi AM, Mansoori MN, Srivastava K, Raghuvanshi A, Maurya R, Trivedi R, Goel A, Singh D (2014) miR-542-3p suppresses osteoblast cell proliferation and differentiation, targets BMP-7 signaling and inhibits bone formation. Cell Death Dis 5:e1050. https://doi.org/10.1038/cddis.2014.4 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Bhargavan B, Singh D, Gautam AK, Mishra JS, Kumar A, Goel A, Dixit M, Pandey R, Manickavasagam L, Dwivedi SD, Chakravarti B, Jain GK, Ramachandran R, Maurya R, Trivedi A, Chattopadhyay N, Sanyal S (2012) Medicarpin, a legume phytoalexin, stimulates osteoblast differentiation and promotes peak bone mass achievement in rats: evidence for estrogen receptor beta-mediated osteogenic action of medicarpin. J Nutr Biochem 23:27–38CrossRefPubMedGoogle Scholar
- 15.Tyagi AM, Gautam AK, Kumar A, Srivastava K, Bhargavan B, Trivedi R, Saravanan S, Yadav DK, Singh N, Pollet C, Brazier M, Mentaverri R, Maurya R, Chattopadhyay N, Goel A, Singh D (2010) Medicarpin inhibits osteoclastogenesis and has nonestrogenic bone conserving effect in ovariectomized mice. Mol Cell Endocrinol 325:101–109CrossRefPubMedGoogle Scholar
- 31.Johnson JA, Hemnes AR, Perrien DS, Schuster M, Robinson LJ, Gladson S, Loibner H, Bai S, Blackwell TR, Tada Y, Harral JW, Talati M, Lane KB, Fagan KA, West J (2012) Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension. American Journal of Physiology Lung Cellular and Molecular Physiology 302:L474–L484CrossRefPubMedGoogle Scholar
- 32.Bhargavan B, Gautam AK, Singh D, Kumar A, Chaurasia S, Tyagi AM, Yadav DK, Mishra JS, Singh AB, Sanyal S, Goel A, Maurya R, Chattopadhyay N (2009) Methoxylated isoflavones, cajanin and isoformononetin, have non-estrogenic bone forming effect via differential mitogen activated protein kinase (MAPK) signaling. J Cell Biochem 108:388–399CrossRefPubMedGoogle Scholar
- 33.Gautam AK, Bhargavan B, Tyagi AM, Srivastava K, Yadav DK, Kumar M, Singh A, Mishra JS, Singh AB, Sanyal S, Maurya R, Manickavasagam L, Singh SP, Wahajuddin W, Jain GK, Chattopadhyay N, Singh D (2011) Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats. J Nutr Biochem 22:318–327CrossRefPubMedGoogle Scholar
- 34.Pandey R, Gautam AK, Bhargavan B, Trivedi R, Swarnkar G, Nagar GK, Yadav DK, Kumar M, Rawat P, Manickavasagam L, Kumar A, Maurya R, Goel A, Jain GK, Chattopadhyay N, Singh D (2010) Total extract and standardized fraction from the stem bark of Butea monosperma have osteoprotective action: evidence for the nonestrogenic osteogenic effect of the standardized fraction. Menopause 17:602–610PubMedGoogle Scholar
- 35.Tyagi AM, Mansoori MN, Srivastava K, Khan MP, Kureel J, Dixit M, Shukla P, Trivedi R, Chattopadhyay N, Singh D (2014) Enhanced immunoprotective effects by anti-IL-17 antibody translates to improved skeletal parameters under estrogen deficiency compared with anti-RANKL and anti-TNF-alpha antibodies. Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research 29:1981–1992CrossRefGoogle Scholar
- 36.Tyagi AM, Srivastava K, Mansoori MN, Trivedi R, Chattopadhyay N, Singh D (2012) Estrogen deficiency induces the differentiation of IL-17 secreting Th17 cells: a new candidate in the pathogenesis of osteoporosis. PLoS One 7:e44552. https://doi.org/10.1371/journal.pone.0044552 CrossRefPubMedPubMedCentralGoogle Scholar