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Mesenchymal stromal cells (MSCs) are used to treat infectious and immune diseases and disorders; however, its mechanism(s) remain incompletely defined. Here we find that bone marrow stromal cells (BMSCs) lacking Pinch1/2 proteins display dramatically reduced ability to suppress lipopolysaccharide (LPS)-induced acute lung injury and dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice. Prx1-Cre; Pinch1 f/f ; Pinch2 -/- transgenic mice have severe defects in both immune and hematopoietic functions, resulting in premature death, which can be restored by intravenous injection of wild-type BMSCs. Single cell sequencing analyses reveal dramatic alterations in subpopulations of the BMSCs in Pinch mutant mice. Pinch loss in Prx1 + cells blocks differentiation and maturation of hematopoietic cells in the bone marrow and increases production of pro-inflammatory cytokines TNF-α and IL-1β in monocytes. We find that Pinch is critical for expression of Cxcl12 in BMSCs; reduced production of Cxcl12 protein from Pinch-deficient BMSCs reduces expression of the Mbl2 complement in hepatocytes, thus impairing the innate immunity and thereby contributing to infection and death. Administration of recombinant Mbl2 protein restores the lethality induced by Pinch loss in mice. Collectively, we demonstrate that the novel Pinch-Cxcl12-Mbl2 signaling pathway promotes the interactions between bone and liver to modulate immunity and hematopoiesis and may provide a useful therapeutic target for immune and infectious diseases., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Osteocytes act as mechanosensors in bone; however, the underlying mechanism remains poorly understood. Here we report that deleting Kindlin-2 in osteocytes causes severe osteopenia and mechanical property defects in weight-bearing long bones, but not in non-weight-bearing calvariae. Kindlin-2 loss in osteocytes impairs skeletal responses to mechanical stimulation in long bones. Control and cKO mice display similar bone loss induced by unloading. However, unlike control mice, cKO mice fail to restore lost bone after reloading. Osteocyte Kindlin-2 deletion impairs focal adhesion (FA) formation, cytoskeleton organization and cell orientation in vitro and in bone. Fluid shear stress dose-dependently increases Kindlin-2 expression and decreases that of Sclerostin by downregulating Smad2/3 in osteocytes; this latter response is abolished by Kindlin-2 ablation. Kindlin-2-deficient osteocytes express abundant Sclerostin, contributing to bone loss in cKO mice. Collectively, we demonstrate an indispensable novel role of Kindlin-2 in maintaining skeletal responses to mechanical stimulation by inhibiting Sclerostin expression during osteocyte mechanotransduction.

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.

Mammalian focal adhesion proteins Pinch1 and Pinch2 regulate integrin activation and cell-extracellular matrix adhesion and migration. Here, we show that deleting Pinch1 in osteocytes and mature osteoblasts using the 10-kb mouse Dmp1-Cre and Pinch2 globally (double KO; dKO) results in severe osteopenia throughout life, while ablating either gene does not cause bone loss, suggesting a functional redundancy of both factors in bone. Pinch deletion in osteocytes and mature osteoblasts generates signals that inhibit osteoblast and bone formation. Pinch-deficient osteocytes and conditioned media from dKO bone slice cultures contain abundant sclerostin protein and potently suppress osteoblast differentiation in primary BM stromal cells (BMSC) and calvarial cultures. Pinch deletion increases adiposity in the BM cavity. Primary dKO BMSC cultures display decreased osteoblastic but enhanced adipogenic, differentiation capacity. Pinch loss decreases expression of integrin β3, integrin-linked kinase (ILK), and α-parvin and increases that of active caspase-3 and -8 in osteocytes. Pinch loss increases osteocyte apoptosis in vitro and in bone. Pinch loss upregulates expression of both Rankl and Opg in the cortical bone and does not increase osteoclast formation and bone resorption. Finally, Pinch ablation exacerbates hindlimb unloading-induced bone loss and impairs active ulna loading-stimulated bone formation. Thus, we establish a critical role of Pinch in control of bone homeostasis.