Pyk2 deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene

Abstract

Bone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17β-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor β (ERβ). As a consequence, E2 signaling via ERβ was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERβ signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases.

Short-term pharmacologic RAGE inhibition differentially affects bone and skeletal muscle in middle-aged mice

“…Adherent-BMCs were cultured for 10 days in α-MEM supplemented with 10% FBS and 1% penicillin/streptomycin in the presence of 50 μM ascorbic acid and 10 mM β-glycerol phosphate, as previously described (Posritong et al, 2018). Cells were fixed with 3.7% paraformaldehyde in PBS for 1 hour, rinsed twice with distilled water, and stained for 10 min with 40 mM Alizarin Red S (pH 4.2) at room temperature under shaking.…”

Proline-Rich Protein Tyrosine Kinase 2 in Inflammation and Cancer

“…In eosinophils, Pyk2 is not required for integrin-mediated adhesion, but is essential for β2 integrin-mediated spreading and migration [ 28 ]. Pyk2 is involved in normal bone remodeling [ 29 , 30 , 31 ]. Pyk2 −/− mice exhibit increased bone mass due to increased bone formation, resulted from enhanced osteoprogenitor cell differentiation and activation [ 31 ].…”

A Pyk2 inhibitor incorporated into a PEGDA-gelatin hydrogel promotes osteoblast activity and mineral deposition

“…We and others have shown that Pyk2 genetic deletion leads to increased bone bass, through both increased bone formation by osteoblasts and decreased bone-degradation by osteoclasts [16,19]. Further, we reported that Pyk2-deficient osteoblasts show increased proliferation and mineral deposition than wild-type (WT) osteoblasts, and that the osteogenic effect of Pyk2deletion was associated with the estrogen signaling cascade [20]. Further, it was shown that ovariectomized rats treated with the chemical inhibitor PF-431396 (PF-43), which targets both Pyk2 and the related protein FAK, had increased bone formation and were protected from ovariectomy-induced bone loss [16].…”