Regulator of G Protein Signaling Protein 12 (Rgs12) Controls Mouse Osteoblast Differentiation via Calcium Channel/Oscillation and Gαi-ERK Signaling

Bone homeostasis intimately relies on the balance between osteoblasts (OBs) and osteoclasts (OCs). Our previous studies have revealed that regulator of G protein signaling protein 12 (Rgs12), the largest protein in the Rgs super family, is essential for osteoclastogenesis from hematopoietic cells and OC precursors. However, how Rgs12 regulates OB differentiation and function is still unknown. To understand that, we generated an OB-targeted Rgs12 conditional knockout (CKO) mice model by crossing Rgs12 fl/fl mice with Osterix (Osx)-Cre transgenic mice. We found that Rgs12 was highly expressed in both OB precursor cells (OPCs) and OBs of wild-type (WT) mice, and gradually increased during OB differentiation, whereas Rgs12-CKO mice (Osx Cre/+ ; Rgs12 fl/fl ) exhibited a dramatic decrease in both trabecular and cortical bone mass, with reduced numbers of OBs and increased apoptotic cell population. Loss of Rgs12 in OPCs in vitro significantly inhibited OB differentiation and the expression of OB marker genes, resulting in suppression of OB maturation and mineralization. Further mechanism study showed that deletion of Rgs12 in OPCs significantly inhibited guanosine triphosphatase (GTPase) activity and cyclic adenosine monophosphate (cAMP) level, and impaired Calcium (Ca 2+ ) oscillations via restraints of major Ca 2+ entry sources (extracellular Ca 2+ influx and intracellular Ca 2+ release from endoplasmic reticulum), partially contributed by the blockage of L-type Ca 2+ channel mediated Ca 2+ influx. Downstream mediator extracellular signal-related protein kinase (ERK) was found inactive in OBs of Osx Cre/+ ; Rgs12 fl/fl mice and in OPCs after Rgs12 deletion, whereas application of pertussis toxin (PTX) or overexpression of Rgs12 could rescue the defective OB differentiation via restoration of ERK phosphorylation. Our findings reveal that Rgs12 is an important regulator during osteogenesis and highlight Rgs12 as a potential therapeutic target for bone disorders. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Researc

TIEG1/KLF10 Modulates Runx2 Expression and Activity in Osteoblasts

Deletion of TIEG1/KLF10 in mice results in a gender specific osteopenic skeletal phenotype with significant defects in both cortical and trabecular bone, which are observed only in female animals. Calvarial osteoblasts isolated from TIEG1 knockout (KO) mice display reduced expression levels of multiple bone related genes, including Runx2, and exhibit significant delays in their mineralization rates relative to wildtype controls. These data suggest that TIEG1 plays an important role in regulating Runx2 expression in bone and that decreased Runx2 expression in TIEG1 KO mice is in part responsible for the observed osteopenic phenotype. In this manuscript, data is presented demonstrating that over-expression of TIEG1 results in increased expression of Runx2 while repression of TIEG1 results in suppression of Runx2. Transient transfection and chromatin immunoprecipitation assays reveal that TIEG1 directly binds to and activates the Runx2 promoter. The zinc finger containing domain of TIEG1 is necessary for this regulation supporting that activation occurs through direct DNA binding. A role for the ubiquitin/proteasome pathway in fine tuning the regulation of Runx2 expression by TIEG1 is also implicated in this study. Additionally, the regulation of Runx2 expression by cytokines such as TGFβ1 and BMP2 is shown to be inhibited in the absence of TIEG1. Co-immunoprecipitation and co-localization assays indicate that TIEG1 protein associates with Runx2 protein resulting in co-activation of Runx2 transcriptional activity. Lastly, Runx2 adenoviral infection of TIEG1 KO calvarial osteoblasts leads to increased expression of Runx2 and enhancement of their ability to differentiate and mineralize in culture. Taken together, these data implicate an important role for TIEG1 in regulating the expression and activity of Runx2 in osteoblasts and suggest that decreased expression of Runx2 in TIEG1 KO mice contributes to the observed osteopenic bone phenotype

Building bone to reverse osteoporosis and repair fractures

An important, unfilled clinical need is the development of new approaches to improve fracture healing and to treat osteoporosis by increasing bone mass. Recombinant forms of bone morphogenetic protein 2 (BMP2) and BMP7 are FDA approved to promote spinal fusion and fracture healing, respectively, and the first FDA-approved anabolic drug for osteoporosis, parathyroid hormone, increases bone mass when administered intermittently but can only be given to patients in the US for two years. As we discuss here, the tremendous explosion over the last two decades in our fundamental understanding of the mechanisms of bone remodeling has led to the prospect of mechanism-based anabolic therapies for bone disorders

MicroRNA Functions in Osteogenesis and Dysfunctions in Osteoporosis

MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression that control osteoblast mediated bone formation and osteoclast-related bone remodeling. Deregulation of miRNA mediated mechanisms is emerging as an important pathological factor in bone degeneration (eg, osteoporosis) and other bone-related diseases. MiRNAs are intriguing regulatory molecules that are networked with cell signaling pathways and intricate transcriptional programs through ingenuous circuits with remarkably simple logic. This overview examines key principles by which miRNAs control differentiation of osteoblasts as they evolve from mesenchymal stromal cells during osteogenesis, or of osteoclasts as they originate from monocytic precursors in the hematopoietic lineage during osteoclastogenesis. Of particular note are miRNAs that are temporally upregulated during osteoblastogenesis (eg, miR-218) or osteoclastogenesis (eg, miR-148a). Each miRNA stimulates differentiation by suppressing inhibitory signaling pathways ('double-negative' regulation). The excitement surrounding miRNAs in bone biology stems from the prominent effects that individual miRNAs can have on biological transitions during differentiation of skeletal cells and correlations of miRNA dysfunction with bone diseases. MiRNAs have significant clinical potential which is reflected by their versatility as disease-specific biomarkers and their promise as therapeutic agents to ameliorate or reverse bone tissue degeneration

TIEG1 Null Mouse-Derived Osteoblasts Are Defective in Mineralization and in Support of Osteoclast Differentiation In Vitro

Transforming growth factor β-inducible early gene 1 (TIEG1) is a member of the Krüppel-like transcription factor family. To understand the physiological role of TIEG1, we generated TIEG(−/−) (null) mice and found that the TIEG(−/−) mice had increased osteoblast numbers with no increased bone formation parameters. However, when calvarial osteoblasts (OBs) were isolated from neonatal TIEG(−/−) and TIEG(+/+) mice and cultured in vitro, the TIEG(−/−) cells displayed reduced expression of important OB differentiation markers. When the OBs were differentiated in vitro by treatment with bone morphogenic protein 2, the OBs from TIEG(+/+) calvaria displayed several mineralized nodules in culture, whereas those from TIEG(−/−) mice showed no nodules. To characterize the OBs' ability to support osteoclast differentiation, the OBs from TIEG(+/+) and TIEG(−/−) mice were cultured with marrow and spleen cells from TIEG(+/+) mice. Significantly fewer osteoclasts developed when TIEG(−/−) OBs were used to support osteoclast differentiation than when TIEG(+/+) OBs were used. Examination of gene expression in the TIEG(−/−) OBs revealed decreased RANKL and increased OPG expression compared to TIEG(+/+) OBs. The addition of RANKL to these cocultures only partially restored the ability of TIEG(−/−) OBs to support osteoclast differentiation, whereas M-CSF alone or combined with RANKL had no additional effect on osteoclast differentiation. We conclude from these data that TIEG1 expression in OBs is critical for both osteoblast-mediated mineralization and osteoblast support of osteoclast differentiation

TGF-β coordinately activates TAK1/MEK/AKT/NFkB and SMAD pathways to promote osteoclast survival

To better understand the roles of TGF-β in bone metabolism, we investigated osteoclast survival in response TGF-β and found that TGF-β inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-β receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-β treatment. Since osteoclast survival involves MEK, AKT, and NFkappaB activation, we examined TGF-β effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IkappaB, and NFkappaB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFkappaB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFkappaB repressed TGF-β-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-β-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclX(L) expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-β-induced NFkappaB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-β to support of osteoclast survival

Model depicting the mechanisms by which TIEG1 mediates Runx2 expression and activity in osteoblasts.

<p>Model depicting the mechanisms by which TIEG1 mediates Runx2 expression and activity in osteoblasts.</p