Mechanical forces are particularly effective at activating mitogen-activated protein kinase (MAPK) cascades in nearly all types of bone cells evaluated. MAPKs are serine/threonine protein kinases necessary for bone cell differentiation, proliferation, and survival. In endothelial cells within bone, mechanical factors activate not only extracellular signal-related kinases 1 and 2 (ERK1/2) but also p38, beta-cell myeloid kinase 1 (BMK-1), and c-Jun terminal kinases (JNK). Mechanical activation of ERK1/2 has been shown to be critical for certain strain responses in bone stromal and osteoblastic cells [3]. ERK1/2 causes downregulation of receptor activator of nuclear factor kappa-B ligand (RANKL) and upregulation of endothelial nitric oxide synthase (eNOS) protein after bone cells are exposed to strain. Activation of these pathways leads to decreased bone resorption and increased bone formation.

Activation of ERK1/2 is also necessary for transforming growth factor-β (TGFβ)-induced osteogenic differentiation of mesenchymal stem cells (MSCs) into preosteoblasts and osteoblasts. ERK1/2 activation in bone cells also occurs during mechanical stimulation of voltage-sensitive calcium channels (VSCCs). Activation of ERK1/2 in osteoblastic cells by fluid shear requires Ca²⁺ influx through VSCCs and is ATP dependent. Osteocyte-like MLO-Y4 cells require association of the α₂δ₁ auxiliary subunit with VSCCs in order for mechanical signals to activate ERK1/2.

Protein kinase B (Akt) is a serine/threonine kinase involved in a broad range of cellular functions that is activated by a variety of growth factors, cytokines, and mechanical signals [4]. Mechanical signals that activate Akt lead to increased β-catenin activity, which causes inhibition of MSC adipogenesis, thereby causing differentiation of bone marrow MSCs toward the osteoblast lineage. Exposure to mechanical strain leading to Akt activation results in increased focal adhesion assembly in bone cells. Skeletal loading resulting in increased bone strength is influenced by refractory periods that allow regeneration of enzymes, recycling of molecules to the cell surface, and the numbers and arrangement of cytoskeletal platforms initiating the signaling cascade.

Focal adhesion kinase (FAK) is a non-receptor cytoplasmic protein tyrosine kinase (PTK) that is found in higher concentrations near focal adhesions between cells. This kinase plays an important role in signaling events involving growth factors, extracellular matrix (ECM) molecules, and stress signals [5]. FAK is found in proximity to signaling proteins including sarcoma gene (Src) family PTKs, phosphatidylinositol-3-kinases (PI3K), and paxillin. Interactions with these associated signaling proteins enable FAK to form a functional network of integrin-stimulated signaling pathways that result in activation of downstream targets, including MAPK pathways. Activation of FAK leads to autophosphorylation of FAK tyrosine 397, which leads to interactions with Src-family proteins and other molecules containing Src homology 2 (SH2) domains. Phosphorylation of FAK leads to MAPK activation by interacting with chicken sarcoma gene (c-Src), growth factor receptor-bound protein 2 (Grb2), and rat sarcoma gene (Ras), explaining why most biophysical stimuli in cell culture cause activation of MAPK pathways. Oscillatory fluid flow (OFF) has been demonstrated to cause sustained association of Src and FAK with α_vβ₃ integrin. Stimulation of FAK caused by this integrin association upregulates PI3K activity and modulates downstream ERK and Akt/mammalian target of rapamycin (mTOR)/p70S6K pathways, leading to increased osteoblast proliferation. FAK has also been shown to contribute to OFF-induced stimulation of osteopontin (OPN) and cyclooxygenase-2 (COX-2) expression in osteoblasts. This effect is critical for fluid shear stress-mediated increased expression of osteocalcin (OCN), core-binding factor subunit α1 (Runx2), and Osterix (Osx), demonstrating that FAK is important for osteoblast differentiation and bone formation.

β-Catenin is critical for bone formation by osteoblasts, but plays other roles in bone biology. A mutation in the Wingless (Wnt) co-receptor lipid-related protein receptor-5/6 (LRP5/6) that causes constitutive activation of Wnt stimulates bone formation leading to a high bone mass phenotype known as sclerosteosis, and a dominant negative mutation of the same LRP5/6 receptor results in a low bone mass phenotype known as osteoporosis-pseudoglioma syndrome. Multiple studies have demonstrated the role of β-catenin in osteoblasts and osteocytes. β-Catenin regulates both bone formation and resorption [6, 7]. Skeletal loading has been shown to regulate levels of β-catenin in bone cells in animals and humans.

While the LRP5/6 receptor may function as a mechanoreceptor in osteoblasts, mechanical strain has been shown to control MSC differentiation via non-LRP5/6 regulation of β-catenin. Two percent mechanical strain at 10 cycles/min and continuous fluid flow at 8 dyn/cm² and OFF at 10 dyn/cm² have each been shown to stimulate β-catenin activity in osteoblasts in spite of blocked LRP5/6 receptors. Low-intensity vibration at <10με and 90 cycles per second stimulates β-catenin and suppresses fat cell differentiation, indicating that both high- and low-magnitude mechanical stimuli alter MSC fate through β-catenin. Focal adhesion-based connections with substrate mediate glycogen synthase kinase-3β (GSK3β) inhibition after loading of MSCs and osteoblasts, resulting in increased β-catenin due to reduced proteosomal degradation. Proximal signaling results in mechanical activation of mTORC2, leading to phosphorylation of Akt serine 473, leading to Akt-dependent inhibition of GSK3β (Fig. 2.2). If mTORC2 functions as a mechanical target, there may be interactions between the cytoskeleton and metabolic responses to exercise since mTORC2 responds to insulin signaling.

Guanosine triphosphatases (GTPases) are a large family of enzymes that bind to and hydrolyze GTP. These enzymes function as switches regulating a wide variety of physiological processes. Mechanical stimuli activate heterotrimeric GTPases via G-protein-coupled receptors, resulting in increased intracellular calcium ([Ca²⁺]_i), cyclic AMP (cAMP), and cyclic GMP (cGMP) (Fig. 2.3). Nitric oxide (NO) is produced by protein kinase G (PKG) after mechanical shear stress in osteoblasts [8]. PKGII was shown to be necessary for Src upregulation by phosphorylating Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1). Subsequent fluid shear stress led to recruitment of PKGII, Src, SHP-1, and SHP-2 to a β3-integrin-containing mechanosome.

Estrogen deficiency after menopause is a major cause of osteoporosis, and estrogen receptors (ERs) play a key role in postmenopausal bone loss. ERα mediates bone formation in response to load-bearing in vivo and regulates how osteoblasts and osteocytes respond to mechanical stimulation [9]. ER also modulates mechanically activated signaling pathways. ERα knockout (KO) mice were shown to have reduced response to tibial loading, with severely reduced and delayed transcriptional response and a broad range of effects compared to wild-type littermates. Three hours following a brief loading regimen, wild-type mouse tibiae showed altered transcription of 642 genes, while only 26 genes were modified in ERα KO tibiae. For example, sclerostin gene (SOST) expression was significantly reduced after loading in wild-type mice, but unchanged in loaded tibiae of ERα KO mice.

Bone cells respond to mechanical stimuli via ERα through both genomic and nongenomic actions. ERα nongenomic actions depend on direct interaction with insulin-like growth factor-1 receptor (IGF-1R), leading to sensitization of IGF-1R and upregulation of early strain-regulated genes including COX-2. β1-Integrin expression, which is important for load-induced bone formation, is also upregulated by ERα. These interactions help explain why estrogen may help upregulate osteogenic target genes in response to mechanical stimuli.

A rapid increase in [Ca²⁺]_i is the earliest detectable response in mechanically activated bone cells [10]. Calcium channels in the plasma membrane and intracellular organelles help regulate [Ca²⁺]_i. [Ca²⁺]_i concentrations are tightly regulated to maintain a very low level of free [Ca²⁺]_i, making [Ca²⁺]_i an excellent second messenger system. [Ca²⁺]_i serves as an initial signal in bone cell proliferation, mitosis, differentiation, and motility. [Ca²⁺]_i mobilization is initiated by cell membrane strain, pressure, fluid flow, and osmotic swelling. The frequency of [Ca²⁺]_i spiking is more important in bone cell response to loading than the magnitude of Ca²⁺ spikes, and a rest period between loading enhances Ca²⁺ response in osteoblastic cells.

Changes in [Ca²⁺]_i are linked to several mechanically regulated signaling cascades, including inositol-3-phosphate (IP3), adenosine triphosphate (ATP), and NO. [Ca²⁺]_i mobilization stimulates downstream signaling through protein kinase A (PKA), MAPK, and c-Fos. Prostaglandin E₂ (PGE₂) release plays an important role in mechanical bone formation, but is activated by a Ca²⁺-independent mechanism. Other studies show that PGE₂ release is dependent on Ca²⁺ entry through the L-type VSCC, followed by release of ATP.

Mechanical strain applied to mesenchymal stem cells causes these cells to differentiate toward osteoblasts and suppresses differentiation toward adipocytes [11]. Bone formation occurs during embryonic growth and development and during postnatal bone modeling, remodeling, and repair. In order for osteoblasts to continue to fulfill their role, the skeleton or other tissues must contain a sufficiently large reservoir of osteoprogenitor cells to generate new osteoblasts from birth until death. Osteoprogenitor cells maintain their ability to differentiate from a less differentiated precursor state, or to transdifferentiate from other differentiated cell types, into osteoblast-like cells that produce a variety of proteins characterizing osteoblasts, such as Runx2, alkaline phosphatase (ALP), ON, and OCN, and synthesize bone matrix. Because osteoprogenitor cells have not yet been identified to express specific protein markers, they are difficult to identify, and the precise anatomical location of their niche remains unknown. Osteoprogenitor subpopulations have been identified in bone marrow, but also in cardiovascular tissues, including the aortic heart valve, vascular smooth muscle, and capillary beds. Mechanical signals are also thought to regulate the function and differentiation of osteoprogenitor cells in tissues other than the bone.