UC San Diego

Skeletal integrity is preserved by continuous bone remodeling by osteoblasts and osteoclasts. While the careful regulation between bone formation and resorption is known to be a hallmark of bone maintenance, the signaling mechanisms which drive each of these processes are incompletely understood. Estrogens and fluid shear stress are known to exert positive effects on osteoblast proliferation and osteocyte survival. Both stimuli have been shown to rapidly increase nitric oxide (NO) production in a number of cell types, suggesting an activation of the NO/cGMP/PKG pathway. NO is involved in regulating cell survival and proliferation in different cell types. In the first part of the dissertation, we show that estrogen-mediated osteocytes survival is regulated through the NO/cGMP pathway. Trypan blue, TUNEL, and cleaved caspase 3 staining show the inability of estrogen to protect osteocytes from etoposide-induced apoptosis in the presence of pharmacological inhibitors of NOS, soluble guanylate cyclase, and PKG, or siRNA targeting PKG. The pro-survival effects of estrogen were mimicked by a membrane-permeable analog of cGMP. We show that PKGs function in a dual mechanism to prevent apoptosis; PKGII activates Akt and ERK, and PKGI[alpha] directly phosphorylates the Bcl-2 family member, BAD. cGMP is also involved in regulating mRNA expression of pro-apoptotic genes. The second part of this dissertation focuses on the role of PKGII in mechanotransduction. We first analyzed the effects of the NO/cGMP pathway on downstream fos family gene expression. We determined that fos genes were induced upon fluid shear stress through a NO/cGMP/PKGII mechanism activating MEK and ERK. We found that PKGII is recruited to a complex containing SHP-1, SHP-2, Src, and [alpha]v[Beta]₃ integrins in response to fluid shear stress. SHP-1 is directly phosphorylated by PKGII, and in turn, dephosphorylates and activates Src, allowing for downstream ERK signaling. In conclusion, we reveal a integral role for NO/cGMP/PKG signaling in the maintenance of bone integrity