Osteoarthritis is a chronic, debilitating disease that causes long-term pain and immobility. Germline deletion of Phlpp1 or administration of small molecules that inhibit Phlpp1 prevents post-traumatic osteoarthritis (PTOA) in mice. However, the chondrocyte-intrinsic role of Phlpp1 in PTOA progression is unknown. The objective of this study was to determine how postnatal, chondrocyte-directed deletion of Phlpp1 affects PTOA progression in the presence or absence of Phlpp inhibitors. Phlpp1^fl/fl; Agc-Cre^ERT2 and Agc-Cre^ERT2 mice were injected with tamoxifen at 12 weeks of age to generate Phlpp1-CKO_AgcERT and control (AgcERT) groups. Male mice underwent surgery to destabilize the medial meniscus (DMM) at 17 weeks of age. A separate cohort of male Phlpp1-CKO_AgcERT mice were administered an intra-articular injection of NSC117079, a Phlpp1/2 inhibitor, or saline seven weeks after DMM surgery. Activity and mechanical allodynia were monitored throughout the experiment and cartilage damage was evaluated 12 weeks post-surgery. Phlpp1-CKO_AgcERT mice had less cartilage damage than AgcERT littermates 12 weeks after DMM surgery but exhibited no differences in activity. Prg4 expression was also higher in articular chondrocytes of Phlpp1-CKO_AgcERT mice. Intra-articular administration of NSC117079 to Phlpp1-CKO_AgcERT mice improved cartilage structure, subchondral bone sclerosis, and mechanical allodynia at 12 weeks post-DMM. Postnatal deletion of Phlpp1 in chondrocytes attenuates DMM-induced cartilage damage and subchondral bone sclerosis but does not prevent pain-related behaviors. Intra-articular injection of Phlpp inhibitors delays mechanical allodynia in Phlpp1-CKO_AgcERT mice. These data indicate that Phlpp1 in chondrocytes affects articular cartilage structure after injury, but pain-related behaviors are controlled by Phlpp1 or Phlpp2 in other cell types.

Osteoporosis and other metabolic bone diseases are prevalent in the aging population. While bone has the capacity to regenerate throughout life, bone formation rates decline with age and contribute to reduced bone density and strength. Identifying mechanisms and pathways that increase bone accrual in adults could prevent fractures and accelerate healing. G protein-gated inwardly rectifying K⁺ (GIRK) channels are key effectors of G protein-coupled receptor signaling. Girk3 was recently shown to regulate endochondral ossification. Here, we demonstrate that deletion of Girk3 increases bone mass after 18 weeks of age. Male 24-week-old Girk3^-/- mice have greater trabecular bone mineral density and bone volume fraction than wildtype (WT) mice. Osteoblast activity is moderately increased in 24-week-old Girk3^-/- mice compared to WT mice. In vitro, Girk3^-/- bone marrow stromal cells (BMSCs) are more proliferative than WT BMSCs. Calvarial osteoblasts and BMSCs from Girk3^-/- mice are also more osteogenic than WT cells, with altered expression of genes that regulate the wingless-related integration site (Wnt) family. Wnt inhibition via Dickkopf-1 (Dkk1) or β-catenin inhibition via XAV939 prevents enhanced mineralization, but not proliferation, in Girk3^-/- BMSCs and slows these processes in WT cells. Finally, selective ablation of Girk3 from cells expressing Cre recombinase from the 2.3 kb-Col1a1 promoter, including osteoblasts and osteocytes, is sufficient to increase bone mass and bone strength in male mice at 24 weeks of age. Taken together, these data demonstrate that Girk3 regulates progenitor cell proliferation, osteoblast differentiation, and bone mass accrual in adult male mice. Osteoporosis is a prevalent, debilitating disease characterized by low bone mass. Identifying pathways that enhance bone formation could lead to therapies that prevent fractures and increase bone mass. In this study, we show that whole-body or osteoblast-specific deletion of G protein-gated inwardly rectifying K⁺ channel 3 (Girk3) increases bone mass in male adult mice. Osteoblast progenitor cells from Girk3^-/- animals are more proliferative and differentiate more quickly into mature osteoblasts. Taken together, we show that Girk3 is a novel regulator of bone mass in adult mice.

Germline and osteoblast-directed deletion of G protein-gated inwardly rectifying K+ channel 3 (Girk3) was recently shown to increase bone mass after 18 wk of age in male mice. Here, we show that germline Girk3 deletion also increases trabecular and cortical bone mass and increases the mechanical strength of the femur in female mice after 18 wk of age. Unlike male mice, however, osteoblast-directed Girk3 deletion using 2.3 kb-Col1a1-Cre does not increase bone mass in adult female mice. To discover mechanisms underlying high bone mass in female Girk3−/− mice, bulk RNA-sequencing was performed on 2-d-old calvarial bone, revealing lower expression of proinflammatory cytokines such as IL-1β and IL-6 in Girk3−/− mice. Accordingly, cytokines and chemokines are largely suppressed in the circulation of adult Girk3−/− mice compared to WT littermates. The cytokines GM-CSF, IL-1β, IL-2, and IL-9 are reduced in the serum of both male and female Girk3−/− mice, while eotaxin, IFNγ, MIP-1α, and others are sexually dimorphic. Histomorphometry reveals that osteoclast activity is modestly reduced in Girk3−/− bone, which is supported by in vitro osteoclast resorption assays. However, deletion of Girk3 in myeloid-lineage cells with LysM-Cre is not sufficient to recapitulate high bone mass in either male or female mice. Moreover, female Girk3−/− mice are not protected from ovariectomy-induced bone loss. Finally, single-cell screening using cytometry by time-of-flight in the BM revealed no differences in immune cell abundances due to global Girk3 deletion. Taken together, while Girk3 regulates inflammatory cytokine expression in the bone and serum, deletion of Girk3 in myeloid-lineage cells does not affect bone mass.

Nutritional requirements and physiological responses to foods vary considerably among individuals across the lifespan, reflecting differences in age, metabolic capacity, physical activity level, ge...