LARG GEF and ARHGAP18 orchestrate RhoA activity to control mesenchymal stem cell lineage.

The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cells (MSCs), where adipogenic commitment depletes the available pool for osteogenesis. Cell architecture influences lineage decisions, where interfering with cytoskeletal structure promotes adipogenesis. Mechanical strain suppresses MSC adipogenesis partially through RhoA driven enhancement of cytoskeletal structure. To understand the basis of force-driven RhoA activation, we considered critical GEFs (activators) and GAPs (inactivators) on bone marrow MSC lineage fate. Knockdown of LARG accelerated adipogenesis and repressed basal RhoA activity. Importantly, mechanical activation of RhoA was almost entirely inhibited following LARG depletion, and the ability of strain to inhibit adipogenesis was impaired. Knockdown of ARHGAP18 increased basal RhoA activity and actin stress fiber formation, but did not enhance mechanical strain activation of RhoA. ARHGAP18 null MSCs exhibited suppressed adipogenesis assessed by Oil-Red-O staining and Western blot of adipogenic markers. Furthermore, ARHGAP18 knockdown enhanced osteogenic commitment, confirmed by alkaline phosphatase staining and qPCR of Sp7, Alpl, and Bglap genes. This suggests that ARHGAP18 conveys tonic inhibition of MSC cytoskeletal assembly, returning RhoA to an "off state" and affecting cell lineage in the static state. In contrast, LARG is recruited during dynamic mechanical strain, and is necessary for mechanical suppression of adipogenesis. In summary, mechanical activation of RhoA in mesenchymal progenitors is dependent on LARG, while ARHGAP18 limits RhoA delineated cytoskeletal structure in static cultures. Thus, on and off GTP exchangers work through RhoA to influence MSC fate and responses to static and dynamic physical factors in the microenvironment.

Osteocyte specific responses to soluble and mechanical stimuli in a stem cell derived culture model.

Studying osteocyte behavior in culture has proven difficult because these embedded cells require spatially coordinated interactions with the matrix and surrounding cells to achieve the osteocyte phenotype. Using an easily attainable source of bone marrow mesenchymal stem cells, we generated cells with the osteocyte phenotype within two weeks. These "stem cell derived-osteocytes" (SCD-O) displayed stellate morphology and lacunocanalicular ultrastructure. Osteocytic genes Sost, Dmp1, E11, and Fgf23 were maximally expressed at 15 days and responded to PTH and 1,25(OH)2D3. Production of sclerostin mRNA and protein, within 15 days of culture makes the SCD-O model ideal for elucidating regulatory mechanisms. We found sclerostin to be regulated by mechanical factors, where low intensity vibration significantly reduced Sost expression. Additionally, this model recapitulates sclerostin production in response to osteoactive hormones, as PTH or LIV repressed secretion of sclerostin, significantly impacting Wnt-mediated Axin2 expression, via ß-catenin signaling. In summary, SCD-O cells produce abundant matrix, rapidly attain the osteocyte phenotype, and secrete functional factors including sclerostin under non-immortalized conditions. This culture model enables ex vivo observations of osteocyte behavior while preserving an organ-like environment. Furthermore, as marrow-derived mesenchymal stem cells can be obtained from transgenic animals; our model enables study of genetic control of osteocyte behaviors.

Cell Mechanosensitivity to Extremely Low-Magnitude Signals Is Enabled by a LINCed Nucleus.

A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSCs), high-magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of focal adhesion (FA) kinase (FAK)/mTORC2/Akt signaling generated at FAs. Physiologic systems also rely on a persistent barrage of low-level signals to regulate behavior. Exposing MSC to extremely low-magnitude mechanical signals (LMS) suppresses adipocyte formation despite the virtual absence of substrate strain (<0.001%), suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here, we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating FAK and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the FAs, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by HMS was unaffected by LINC decoupling, consistent with signal initiation at the FA mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with "outside-in" signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent "inside-inside" signaling.

Vibration therapy: clinical applications in bone.

PURPOSE OF REVIEW: The musculoskeletal system is largely regulated through dynamic physical activity and is compromised by cessation of physical loading. There is a need to recreate the anabolic effects of loading on the musculoskeletal system, especially in frail individuals who cannot exercise. Vibration therapy is designed to be a nonpharmacological analogue of physical activity, with an intention to promote bone and muscle strength. RECENT FINDINGS: Animal and human studies suggest that high-frequency, low-magnitude vibration therapy improves bone strength by increasing bone formation and decreasing bone resorption. There is also evidence that vibration therapy is useful in treating sarcopenia, which confounds skeletal fragility and fall risk in aging. Enhancement of skeletal and muscle strength involves regulating the differentiation of mesenchymal stem cells to build these tissues; mesenchymal stem cell lineage allocation is positively promoted by vibration signals. SUMMARY: Vibration therapy may be useful as a primary treatment as well as an adjunct to both physical and pharmacological treatments, but future studies must pay close attention to compliance and dosing patterns, and importantly, the vibration signal, be it low-intensity vibration (<1g) appropriate for treatment of frail individuals or high-intensity vibration (>1g) marketed as a training exercise.

Mechanically activated Fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells.

Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of ß-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation.