Kindlin-2 regulates skeletal homeostasis by modulating PTH1R in mice

Xuekun Fu,Sixiong Lin,Yumei Lai,Guozhi Xiao,Ge Zhang,Sheng Chen,Qinnan Yan,Bo Zhou,Chu Tao,Meiqing Wang,Wenfei Jin,Huiling Cao,Xuenong Zou,Di Chen,Zengwu Shao,Xiaohao Wu,Lei Qin,Youqiang Song

doi:10.1038/s41392-020-00328-y

Abstract

In vertebrates, the type 1 parathyroid hormone receptor (PTH1R) is a critical regulator of skeletal development and homeostasis; however, how it is modulated is incompletely understood. Here we report that deleting Kindlin-2 in osteoblastic cells using the mouse 10-kb Dmp1-Cre largely neutralizes the intermittent PTH-stimulated increasing of bone volume fraction and bone mineral density by impairing both osteoblast and osteoclast formation in murine adult bone. Single-cell profiling reveals that Kindlin-2 loss increases the proportion of osteoblasts, but not mesenchymal stem cells, chondrocytes and fibroblasts, in non-hematopoietic bone marrow cells, with concomitant depletion of osteoblasts on the bone surfaces, especially those stimulated by PTH. Furthermore, haploinsufficiency of Kindlin-2 and Pth1r genes, but not that of either gene, in mice significantly decreases basal and, to a larger extent, PTH-stimulated bone mass, supporting the notion that both factors function in the same genetic pathway. Mechanistically, Kindlin-2 interacts with the C-terminal cytoplasmic domain of PTH1R via aa 474–475 and Gsα. Kindlin-2 loss suppresses PTH induction of cAMP production and CREB phosphorylation in cultured osteoblasts and in bone. Interestingly, PTH promotes Kindlin-2 expression in vitro and in vivo, thus creating a positive feedback regulatory loop. Finally, estrogen deficiency induced by ovariectomy drastically decreases expression of Kindlin-2 protein in osteocytes embedded in the bone matrix and Kindlin-2 loss essentially abolishes the PTH anabolic activity in bone in ovariectomized mice. Thus, we demonstrate that Kindlin-2 functions as an intrinsic component of the PTH1R signaling pathway in osteoblastic cells to regulate bone mass accrual and homeostasis.

Highlights

INTRODUCTION The type1 parathyroid hormone receptor (PTH1R), a G-proteincoupled seven transmembrane receptor (GPCR), is the primary functional receptor for both endogenous PTH and PTH-related protein (PTHrP) ligands
Kindlin-2 interacts with the C-terminal cytoplasmic domain of PTH1R and the stimulatory subunit of G protein, Gsα, in osteoblastic cells or in COS-7 cells overexpressing both factors To explore mechanisms through which Kindlin-2 mediates the PTH actions, we determined whether Kindlin-2 interacts with PTH1R
Estrogen deficiency dramatically reduces Kindlin-2 expression in osteocytes and Kindlin-2 loss completely abolishes intermittent PTH-stimulated bone volume and bone mineral density (BMD) in ovariectomized mice In an effort to identify factors that regulate expression of Kindlin-2 in osteoblastic cells, we found that estrogen significantly increased the level of Kindlin-2 protein in MC-4 cells in a dose-dependent manner (Fig. 6a, b)

Summary

RESULTS

Control and cKO mice with and without PTH treatment. We used the Lineage Cell Depletion Kit combined with flow cytometry to Kindlin-2 loss in osteoblastic cells severely impairs intermittent deplete the hematopoietic cells, such as T cells, B cells, PTH-stimulated increases in bone volume faction and BMD by monocytes/macrophages, granulocytes, and erythrocytes and impairing both osteoblast and osteoclast formation in male and their precursors, from pooled whole BM cells of each group. To investigate whether the proportions of non-hematopoietic cells were affected by Kindlin-2 loss and/or intermittent PTH treatment, we determined the proportions of distal femurs revealed that the PTH-stimulated increases in bone each cell type and cluster for all samples (Fig. 2c and volume and BMD in control mice were dramatically decreased in cKO mice (Fig. 1a–c). Estrogen deficiency dramatically reduces Kindlin-2 expression in osteocytes and Kindlin-2 loss completely abolishes intermittent PTH-stimulated bone volume and BMD in ovariectomized mice In an effort to identify factors that regulate expression of Kindlin-2 in osteoblastic cells, we found that estrogen significantly increased the level of Kindlin-2 protein in MC-4 cells in a dose-dependent manner (Fig. 6a, b). Fulvestrant, an estrogen receptor antagonist, dramatically decreased Kindlin-2 expression in MC-4

DISCUSSION

Findings

MATERIALS AND METHODS