Protein kinase G2 activation restores Wnt signaling and bone mass in glucocorticoid-induced osteoporosis in mice.

Osteoporotic fractures are a major complication of long-term glucocorticoid therapy. Glucocorticoids transiently increase bone resorption, but they predominantly inhibit bone formation and induce osteocyte apoptosis, leading to bone loss. Current treatments of glucocorticoid-induced osteoporosis aim mainly at reducing bone resorption and are, therefore, inadequate. We previously showed that signaling via the NO/cGMP/protein kinase G pathway plays a key role in skeletal homeostasis. Here, we show that pharmacological PKG activation with the guanylyl cyclase-1 activator cinaciguat or expression of a constitutively active, mutant PKG2R242Q restored proliferation, differentiation, and survival of primary mouse osteoblasts exposed to dexamethasone. Cinaciguat treatment of WT mice or osteoblast-specific expression of PKG2R242Q in transgenic mice prevented dexamethasone-induced loss of cortical bone mass and strength. These effects of cinaciguat and PKG2R242Q expression were due to preserved bone formation parameters and osteocyte survival. The basis for PKG2's effects appeared to be through recovery of Wnt/ß-catenin signaling, which was suppressed by glucocorticoids but critical for proliferation, differentiation, and survival of osteoblast-lineage cells. Cinaciguat reduced dexamethasone activation of osteoclasts, but this did not occur in the PKG2R242Q transgenic mice, suggesting a minor role in osteoprotection. We propose that existing PKG-targeting drugs could represent a novel therapeutic approach to prevent glucocorticoid-induced osteoporosis.

Protein Kinase G2 Is Essential for Skeletal Homeostasis and Adaptation to Mechanical Loading in Male but Not Female Mice.

We previously showed that the NO/cGMP/protein kinase G (PKG) signaling pathway positively regulates osteoblast proliferation, differentiation, and survival in vitro, and that cGMP-elevating agents have bone-anabolic effects in mice. Here, we generated mice with an osteoblast-specific (OB) knockout (KO) of type 2 PKG (gene name Prkg2) using a Col1a1(2.3 kb)-Cre driver. Compared to wild type (WT) littermates, 8-week-old male OB Prkg2-KO mice had fewer osteoblasts, reduced bone formation rates, and lower trabecular and cortical bone volumes. Female OB Prkg2-KO littermates showed no bone abnormalities, despite the same degree of PKG2 deficiency in bone. Expression of osteoblast differentiation- and Wnt/ß-catenin-related genes was lower in primary osteoblasts and bones of male KO but not female KO mice compared to WT littermates. Osteoclast parameters were unaffected in both sexes. Since PKG2 is part of a mechano-sensitive complex in osteoblast membranes, we examined its role during mechanical loading. Cyclical compression of the tibia increased cortical thickness and induced mechanosensitive and Wnt/ß-catenin-related genes to a similar extent in male and female WT mice and female OB Prkg2-KO mice, but loading had a minimal effect in male KO mice. We conclude that PKG2 drives bone acquisition and adaptation to mechanical loading via the Wnt/ß-catenin pathway in male mice. The striking sexual dimorphism of OB Prkg2-KO mice suggests that current U.S. Food and Drug Administration-approved cGMP-elevating agents may represent novel effective treatment options for male osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Increased Bone Marrow-Specific Adipogenesis by Clofazimine Causes Impaired Fracture Healing, Osteopenia, and Osteonecrosis Without Extraskeletal Effects in Rats.

Mycobacterium leprae infection causes bone lesions and osteoporosis, however, the effect of antileprosy drugs on the bone is unknown. We, therefore, set out to address it by investigating osteogenic differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs). Out of 7 antileprosy drugs, only clofazimine (CFZ) reduced MSCs viability (IC50 ∼ 1 µM) and their osteogenic differentiation but increased adipogenic differentiation on a par with rosiglitazone, and this effect was blocked by a peroxisome proliferator-activated receptor gamma antagonist, GW9662. CFZ also decreased osteoblast viability and resulted in impaired bone regeneration in a rat femur osteotomy model at one-third human drug dose owing to increased callus adipogenesis as GW9662 prevented this effect. CFZ treatment decreased BM MSC population and homing of MSC to osteotomy site despite drug levels in BM being much less than its in vitro IC50 value. In adult rats, CFZ caused osteopenia in long bones marked by suppressed osteoblast function due to enhanced adipogenesis and increased osteoclast functions. A robust increase in marrow adipose tissue (MAT) by CFZ did not alter the hematologic parameters but likely reduced BM vascular bed leading to osteonecrosis (ON) characterized by empty osteocyte lacunae. However, CFZ had no effect on visceral fat content and was not associated with any metabolic and hematologic changes. Levels of unsaturated fatty acids in MAT were higher than saturated fatty acids and CFZ further increased the former. From these data, we conclude that CFZ has adverse skeletal effects and could be used for creating a rodent ON model devoid of extraskeletal effects.

A nutraceutical composition containing diosmin and hesperidin has osteogenic and anti-resorptive effects and expands the anabolic window of teriparatide.

A combination of diosmin and hesperidin (9:1 ratio) is marketed as a dietary supplement/nutraceutical for cardiovascular health. We studied the skeletal effect of this combination (90% diosmin and 10% hesperidin, henceforth named as DH). We showed that a) in rats with femur osteotomy, DH stimulated callus bone regeneration, b) in growing rats, DH promoted peak bone mass achievement and c) in OVX rats rendered osteopenic, DH completely restored femur trabecular bones and strength along with the increases in surface referent bone formation and serum osteogenic marker. Furthermore, DH suppressed bone resorption in OVX rats as well as in OVX rats treated with teriparatide (human parathyroid hormone 1-34) but did not affect the osteoanabolic effect of teriparatide. These data suggested that DH could prolong the anabolic window of teriparatide. To understand the mechanism of DH action, we performed pharmacokinetic studies and observed that upon its oral administration the only circulating metabolites was diosmetin (the aglycone form of diosmin) while none of the two input flavanones were detectable. Accordingly, subsequent experiments with diosmetin revealed that it was a selective estrogen receptor-ß agonist that stimulated osteoblast differentiation and suppressed sclerostin the anti-osteoblastogenic Wnt antagonist. Taken together, our study defined a positive skeletal effect of DH.

The osteogenic effect of liraglutide involves enhanced mitochondrial biogenesis in osteoblasts.

Liraglutide (Lira), a long-acting glucagon-like peptide 1 receptor (GLP1R) agonist reduces glycosylated hemoglobin in type 2 diabetes mellitus patients. Lira is reported to have bone conserving effect in ovariectomized (OVX) rats. Here, we investigated the osteoanabolic effect of Lira and studied the underlying mechanism. In established osteopenic OVX rats, Lira completely restored bone mass and strength comparable to parathyroid hormone (PTH 1-34). Body mass index normalized bone mineral density of Lira was higher than PTH. The serum levels of osteogenic surrogate pro-collagen type 1 N-terminal pro-peptide (P1NP) and surface referent bone formation parameters were comparable between Lira and PTH. GLP1R, adiponectin receptor 1 (AdipoR1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) levels in bones were downregulated in the OVX group but restored in the Lira group whereas PTH had no effect. In cultured osteoblasts, Lira time-dependently increased GLP1R, AdipoR1 and PGC1α expression. In osteoblasts, Lira rapidly phosphorylated AMP-dependent protein kinase (AMPK), the cellular energy sensor. Exendin 3, a selective GLP1R antagonist and PKA inhibitor H89 blocked Lira-induced increases in osteoblast differentiation, and expression levels of AdipoR1 and PGC1α. Furthermore, H89 inhibited Lira-induced phosphorylation of AMPK and dorsomorphin, an AMPK inhibitor blocked the Lira-induced increases in osteoblast differentiation and AdipoR1 and PGC1α levels. Lira increased mitochondrial number, respiratory proteins and respiration in osteoblasts in vitro and in vivo, and blocking mitochondrial respiration mitigated Lira-induced osteoblast differentiation. Taken together, our data show that Lira has a strong osteoanabolic effect which involves upregulation of mitochondrial function.

Adiponectin signaling and its role in bone metabolism.

Adiponectin, the most prevalent adipo-cytokine in plasma plays critical metabolic and anti-inflammatory roles is fast emerging as an important molecular target for the treatment of metabolic disorders. Adiponectin action is critical in multiple organs including cardio-vascular system, muscle, liver, adipose tissue, brain and bone. Adiponectin signaling in bone has been a topic of active investigation lately. Human association studies and multiple mice models of gene deletion/modification failed to define a clear cause and effect of adiponectin signaling in bone. The most plausible reason could be the multimeric forms of adiponectin that display differential binding to receptors (adipoR1 and adipoR2) with cell-specific receptor variants in bone. Discovery of small molecule agonist of adipoR1 suggested a salutary role of this receptor in bone metabolism. The downstream signaling of adipoR1 in osteoblasts involves stimulation of oxidative phosphorylation leading to increased differentiation via the likely suppression of wnt inhibitor, sclerostin. On the other hand, the inflammation modulatory effect of adiponectin signaling suppresses the RANKL (receptor activator of nuclear factor κ-B ligand) - to - OPG (osteprotegerin) ratio in osteoblasts leading to the suppression of osteoclastogenic response. This review will discuss the adiponectin signaling and its role in skeletal homeostasis and critically assess whether adipoR1 could be a therapeutic target for the treatment of metabolic bone diseases.

The wakefulness promoting drug Modafinil causes adenosine receptor-mediated upregulation of receptor activator of nuclear factor κB ligand in osteoblasts: Negative impact of the drug on peak bone accrual in rats.

Modafinil is primarily prescribed for treatment of narcolepsy and other sleep-associated disorders. However, its off-prescription use as a cognition enhancer increased considerably, specially among youths. Given its increasing use in young adults the effect of modafinil on peak bone accrual is an important issue but has never been investigated. Modafinil treatment to young male rats caused trabecular and cortical bone loss in tibia and femur, and reduction in biomechanical strength. Co-treatment of modafinil with alendronate (a drug that suppresses bone resorption) reversed the trabecular bone loss but failed to prevent cortical loss. Modafinil increased serum type 1 pro-collagen N-terminal protein (P1NP) and collagen type 1 cross-linked C-telopeptide (CTX-1) indicating a high turnover bone loss. The drug also increased receptor activator of nuclear factor κB ligand (RANKL) to osteoprotegerin (OPG) ratio in serum which likely resulted in increased osteoclast number per bone surface. Furthermore, conditioned medium from modafinil treated osteoblasts increased the expression of osteoclastogenic genes in bone marrow-derived macrophages and the effect was blocked by RANKL neutralizing antibody. In primary osteoblasts, modafinil stimulated cAMP production and using pharmacological approach, we showed that modafinil signalled via adenosine receptors (A2AR and A2BR) which resulted in increased RANKL expression. ZM-241,385 (an A2AR inhibitor) and MRS 1754 (an A2BR inhibitor) suppressed modafinil-induced upregulation of RANKL/OPG ratio in the calvarium of new born rat pups. Our data suggests that by activating osteoblast adenosine receptors modafinil increases the production of osteoclastogenic cytokine, RANKL that in turn results in high turnover bone loss in young rats.