Genomic variants within chromosome 14q32.32 regulate bone mass through MARK3 signaling in osteoblasts
Qian Zhang, … , Charles R. Farber, Thomas L. Clemens
Qian Zhang, … , Charles R. Farber, Thomas L. Clemens
Published April 1, 2021
Citation Information: J Clin Invest. 2021;131(7):e142580. https://doi.org/10.1172/JCI142580.
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Research Article Bone biology

Genomic variants within chromosome 14q32.32 regulate bone mass through MARK3 signaling in osteoblasts

Abstract

Bone mineral density (BMD) is a highly heritable predictor of osteoporotic fracture. GWAS have identified hundreds of loci influencing BMD, but few have been functionally analyzed. In this study, we show that SNPs within a BMD locus on chromosome 14q32.32 alter splicing and expression of PAR-1a/microtubule affinity regulating kinase 3 (MARK3), a conserved serine/threonine kinase known to regulate bioenergetics, cell division, and polarity. Mice lacking Mark3 either globally or selectively in osteoblasts have increased bone mass at maturity. RNA profiling from Mark3-deficient osteoblasts suggested changes in the expression of components of the Notch signaling pathway. Mark3-deficient osteoblasts exhibited greater matrix mineralization compared with controls that was accompanied by reduced Jag1/Hes1 expression and diminished downstream JNK signaling. Overexpression of Jag1 in Mark3-deficient osteoblasts both in vitro and in vivo normalized mineralization capacity and bone mass, respectively. Together, these findings reveal a mechanism whereby genetically regulated alterations in Mark3 expression perturb cell signaling in osteoblasts to influence bone mass.

Authors

Qian Zhang, Larry D. Mesner, Gina M. Calabrese, Naomi Dirckx, Zhu Li, Angela Verardo, Qian Yang, Robert J. Tower, Marie-Claude Faugere, Charles R. Farber, Thomas L. Clemens

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Figure 4

Mark3 deletion accelerates osteogenic differentiation in vitro via DVL/JNK/JAG1 pathway.

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Mark3 deletion accelerates osteogenic differentiation in vitro via DVL/...
(A) Mark3 and (B) Oc mRNA levels during osteogenic differentiation at days 0, 7, and 14 in Mark3fl/fl primary osteoblasts transfected with Ad-GFP and Ad-CRE (n = 4). (C) Representative images and quantification of Alizarin red (ARS) staining after 14 days of osteogenic differentiation in Ad-GFP and Ad-Cre transfected primary osteoblasts (n = 3). (D) Mark3 mRNA levels after 48 hours of siRNA treatment in hBMSCs. (E) Representative images and quantification of ARS staining after 14 days of differentiation in control and siRNA transfected primary hBMSCs (n = 3). (F) qRT-PCR analysis of Oc expression after 14 days of osteoblast differentiation (n = 4) in primary hBMSCs. (G) BrdU incorporation for primary osteoblasts isolated from Mark3fl/fl and Mark3fl/fl;Oc-Cre mice (n = 5-6). (H) Western blot to detect cleaved caspase 3 in Ad-GFP– and Ad-Cre–transfected osteoblasts (n = 5). (I) Bulk RNA-Seq of Ad-GFP– and Ad-Cre–transfected Mark3fl/fl primary osteoblasts identifies Notch signaling and cytoskeletal organization as potential pathways regulated by MARK3. (J) Relative mRNA expression of Jag1 and Hes1 at days 0, 7, and 14 of osteogenic differentiation Ad-GFP– and Ad-Cre–transfected Mark3fl/fl osteoblasts (n = 4). (K) Western blot and quantification of JAG1 48 hours after adenovirus treatment of primary osteoblasts (n = 8). (L) Hes1 luciferase reporter assay on Ad-GFP– and Ad-Cre–transfected Mark3fl/fl primary osteoblasts (n = 4–5). (M) Egr1 mRNA levels after 48 hours of Ad-GFP and Ad-Cre transfection (n = 5). (N) Egr1 and Jag1 mRNA after 48 hours of transfection of si-Egr1 in primary osteoblasts (n = 5). (O) Phos-tag SDS-PAGE of DVL to indicate phosphorylated forms of DVL after 48 hours in Ad-GFP– and Ad-Cre–transfected Mark3fl/fl primary osteoblasts (n = 4). (P) Phos-tag SDS-PAGE of DVL to indicate phosphorylated forms of DVL after overexpression of the human MARK3 kinase domain in primary hBMSCs (n = 3). (Q) Immunoblot of p-JNK and t-JNK after 48 hours of Ad-GFP and Ad-Cre transfection (n = 6). Data are represented as mean ± SEM. *P < 0.05, Student’s t test between genotypes.