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We have recently demonstrated that osteopontin (OPN) induces nuclear factor B (NF B)-mediated promatrix metalloproteinase-2 activation through I B␣/ I B␣ kinase (IKK) signaling pathways. However, the molecular mechanism(s) by which OPN regulates promatrix metalloproteinase-9 (pro-MMP-9) activation, MMP-9-dependent cell motility, and tumor growth and the involvement of upstream kinases in regulation of these processes in murine melanoma cells are not well defined. Here we report that OPN induced ␣ v ␤ 3 integrinmediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKK␣/␤ in B16F10 cells. Moreover, NIK was involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induced NIK-dependent NF B activation through ERK/ IKK␣/␤-mediated pathways. Furthermore OPN enhanced NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, cell motility, and tumor growth. Wild type NIK, IKK␣/␤, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKK␣/␤ (dn IKK␣/␤), and dn ERK1/2 suppressed the OPN-induced NF B activation, uPA secretion, pro-MMP-9 activation, cell motility, and chemoinvasion. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. The level of active MMP-9 in the OPN-induced tumor was higher compared with control. To our knowledge, this is the first report that NIK plays a crucial role in OPN-induced NF B activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKK␣/␤-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth.

Mechanical stimulation is crucial for bone growth/remodeling, and fluid shear stress promotes anabolic responses in osteoblasts through multiple second messengers, including nitric oxide (NO), but the underlying mechanisms are not well understood. Here we demonstrate that the NO/cGMP/PKG signaling pathway activates Src in mechanically-stimulated osteoblasts, initiating a proliferative response. PKG II is necessary for Src activation, which also requires Src docking to β3 integrins and Src dephosphorylation by a Shp-1/2 phosphatase complex. PKG II directly phosphorylates and stimulates Shp-1 activity, and fluid shear stress triggers PKG II, Src, and Shp recruitment to a mechanosome containing β3 integrins. PKG II-null mice show defective osteoblast Src/Erk signaling, and decreased Erk-dependent gene expression in bone. Our findings reveal crosstalk between NO/cGMP/PKG and integrin signaling and establish a new mechanism of Src activation. Since Src controls Erk, which is key to osteoblast growth and survival, these results support use of PKG-activating drugs as mechano-mimetics for treating osteoporosis.

Continuous bone remodeling in response to mechanical loading is critical for skeletal integrity, and interstitial fluid flow is an important stimulus for osteoblast/osteocyte growth and differentiation. However, the biochemical signals mediating osteoblast anabolic responses to mechanical stimulation are incompletely understood. In primary human osteoblasts and murine MC3T3-E1 cells, we found that fluid shear stress induced rapid expression of c-fos, fra-1, fra-2, and fosB/⌬fosB mRNAs; these genes encode transcriptional regulators that maintain skeletal integrity. Fluid shear stress increased osteoblast nitric oxide (NO) synthesis, leading to activation of cGMP-dependent protein kinase (PKG). Pharmacological inhibition of the NO/cGMP/PKG signaling pathway blocked shear-induced expression of all four fos family genes. Induction of these genes required signaling through MEK/Erk, and Erk activation was NO/cGMP/PKG-dependent. Treating cells with a membranepermeable cGMP analog partly mimicked the effects of fluid shear stress on Erk activity and fos family gene expression. In cells transfected with small interfering RNAs (siRNA) specific for membrane-bound PKG II, shear-and cGMP-induced Erk activation and fos family gene expression was nearly abolished and could be restored by transducing cells with a virus encoding an siRNA-resistant form of PKG II; in contrast, siRNA-mediated repression of the more abundant cytosolic PKG I isoform was without effect. Thus, we report a novel function for PKG II in osteoblast mechanotransduction, and we propose a model whereby NO/cGMP/PKG II-mediated Erk activation and induction of c-fos, fra-1, fra-2, and fosB/⌬fosB play a key role in the osteoblast anabolic response to mechanical stimulation.Mechanical stress is a primary determinant of bone growth and remodeling; the strength of bone increases with weight bearing and muscular activity and decreases with unloading and disuse (1, 2). Weight bearing and locomotion stimulate interstitial fluid flow through the bone canalicular system, and the resultant shear stress is thought to be a major mechanism whereby mechanical forces stimulate bone growth (1-4). Fluid shear stress activates various signal transduction pathways and initiates an anabolic response in osteocytes and osteoblasts, leading to changes in gene expression and increased cell proliferation and differentiation (1, 5). As part of this response, a rapid and transient increase in intracellular calcium, nitric oxide (NO), 2 and prostaglandin E 2 occurs, and transcription of genes such as c-fos, cox-2, and igf-1/2 is induced (1, 2, 5).The transcription factor complex AP1, composed of Fos and Jun proteins, plays an essential role in bone development and post-natal skeletal homeostasis (6). De-regulated c-fos expression in mice interferes with normal bone development and induces osteosarcomas, whereas c-Fos-deficient mice develop osteopetrosis because of an early arrest in osteoclast differentiation (7, 8). Mice overexpressing Fra-1, Fra-2, or ⌬FosB (a splice variant of FosB) ex...

Background: Estrogens prevent bone loss in part by preventing osteocyte apoptosis. Results: Anti-apoptotic effects of 17␤-estradiol in osteocytes require NO/cGMP-mediated stimulation of Akt and Akt-and cGMP-dependent protein kinase (PKG)-dependent phosphorylation of BAD. Conclusion: PKG types I and II serve independent anti-apoptotic functions in 17␤-estradiol-treated osteocytes, converging on BAD. Significance: These novel mechanisms of 17␤-estradiol-mediated bone protection provide a rationale for developing NO/cGMP-based therapies for osteoporosis.

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Background: Fluid shear increases intracellular calcium and NO/cGMP signaling in osteoblasts. Results: Focal adhesion kinase and protein kinase G are independently activated downstream of calcium; both kinases cooperatively activate Src, leading to Akt/GSK3/␤-catenin signaling in shear-stressed osteoblasts. Conclusion: Osteoblast mechanotransduction requires cross-talk between FAK and PKG to regulate Akt. Significance: cGMP-elevating agents may prove useful for the treatment of osteoporosis.

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