Abstract
The G-protein alpha-subunit G(s)alpha is required for the intracellular cAMP responses to hormones and other agonists. G(s)alpha is known to mediate the cAMP response to parathyroid hormone and other hormones and cytokines in bone and cartilage. To analyze the in vivo role of G(s)alpha signaling in osteoblasts, we developed mice with osteoblast/osteocyte-specific G(s)alpha deficiency (BGsKO) by mating G(s)alpha-floxed mice with collagen Ialpha1 promoter-Cre recombinase transgenic mice. Early skeletal development was normal in BGsKO mice, because formation of the initial cartilage template and bone collar was unaffected. The chondrocytic zones of the growth plates also appeared normal in BGsKO mice. BGsKO mice had a defect in the formation of the primary spongiosa with reduced immature osteoid (new bone formation) and overall length, which led to reduced trabecular bone volume. In contrast, cortical bone was thickened with narrowing of the bone marrow cavity. This was probably due to decreased cortical bone resorption, because osteoclasts were markedly reduced on the endosteal surface of cortical bone. In addition, the expression of alkaline phosphatase, an early osteoblastic differentiation marker, was normal, whereas the expression of the late osteoblast differentiation markers osteopontin and osteocalcin was reduced, suggesting that the number of mature osteoblasts in bone is reduced. Expression of the osteoclast-stimulating factor receptor activator of NF-kappaB ligand was also reduced. Overall, our findings have similarities to parathyroid hormone null mice and confirm that the differential effects of parathyroid hormone on trabecular and cortical bone are primarily mediated via G(s)alpha in osteoblasts. Osteoblast-specific G(s)alpha deficiency leads to reduced bone turnover.
Highlights
Gs␣ 1 is a ubiquitously expressed G-protein ␣-subunit that couples receptors to adenylyl cyclase and is required for recep
Embryonic lethality of germ line Gnas knock-out mice does not allow us to examine the effects of Gs␣ deficiency in skeletal development, which begins later in gestation
In situ hybridization analysis confirmed that collagen I␣1 is expressed in both osteoblasts and osteocytes at similar levels in control and BGsKO
Summary
Gs␣, stimulatory G-protein ␣-subunit; PTH, parathyroid hormone; PTHrP, PTH-related peptide; PPR, PTH/ PTHrP receptor; RANKL, receptor activator of nuclear factor-B ligand; OPG, osteoprotegerin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BGsKO, osteoblast/osteocyte-specific Gs␣ knockout; Col1-Cre, collagen I␣1 promoter-Cre recombinase transgene; TRAP, tartrate-resistant acid phosphatase; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; E, embryonic day. CAMP is an important second messenger in osteoblasts for several hormones and other extracellular factors, such as parathyroid hormone (PTH) and prostaglandin E2 Both PTH and PTH-related peptide (PTHrP), a paracrine regulator of chondrocyte differentiation in growth plates, activate a common receptor (PTH/PTHrP receptor, PPR), which activates Gs␣ as well as other G-proteins [2,3,4]. PTH has been shown to stimulate RANKL and inhibit OPG expression in osteoblasts, which promotes osteoclastic bone resorption [9]. Our findings suggest that osteoblast-specific Gs␣ deficiency leads to reduced bone turnover and that the differential effects of PTH on trabecular and cortical bone are mediated via Gs␣ signaling pathways in osteoblasts
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