Effect of Shear Stress on RhoA Activities and Cytoskeletal Organization in Chondrocytes

Indiana University-Purdue University Indianapolis (IUPUI)Mechanical force environment is a major factor that influences cellular homeostasis and remodeling. The prevailing wisdom in this field demonstrated that a threshold of mechanical forces or deformation was required to affect cell signaling. However, by using a fluorescence resonance energy transfer (FRET)-based approach, we found that C28/I2 chondrocytes exhibited an increase in RhoA activities in response to high shear stress (10 or 20 dyn/cm2), while they showed a decrease in their RhoA activities to intermediate shear stress at 5 dyn/cm2. No changes were observed under low shear stress (2 dyn/ cm2). The observed two-level switch of RhoA activities was closely linked to the shear stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. Collectively, these results herein suggest that intensities of shear stress are critical in differential activation and inhibition of RhoA activities in chondrocytes

The role of mechanical loading in chondrocyte signaling pathways

Chondrocytes are a predominant cell type present in articular cartilage, whose integrity is jeopardized in joint degenerative diseases such as osteoarthritis (OA). In the chondrocytes of patients with OA, the elevated levels of inflammatory cytokines such as interleukin 1β (IL1β) and tumor necrosis factor α (TNFα) have been reported. These cytokines contribute to degradation of cartilage matrix by increasing activities of proteolytic enzymes. In addition to their contribution to proteolytic enzymes, these cytokines adversely affect anabolic activity of chondrocytes by inhibiting the production of proteoglycans and type II collagen. Therefore, blocking the action of these cytokines is a potential strategy to prevent cartilage degradation. Accumulating evidence suggests that mechanical loading contributes to the regulation of cartilage homeostasis. However, the underlying mechanisms are not clear as to how varying magnitudes of mechanical loading trigger differential intracellular signaling pathways at sub-cellular levels, which consequently lead to selective matrix synthesis and degradation. Furthermore, it is not known whether the loading-magnitude dependent responses are linked to degenerative diseases such as OA. Tyrosine kinases such as Src and focal adhesion kinase (FAK) are known to play a crucial role in OA progression. We hypothesized that mechanical loading regulates the sub-cellular activation pattern of Src/FAK, and acts as a suppressor of the OA- or inflammatory cytokine-driven signaling activities. We used live cell imaging approach in conjunction with fluorescence resonance energy transfer (FRET)-based biosensors to investigate real-time molecular events at the sub-cellular level in live chondrocytes. Using two-dimensional (2D) cell culture and shear stress application, we found that Src is activated by inflammatory cytokines (i.e., IL1β and TNFα), and is regulated by shear stress in a magnitude-dependent manner. Importantly, the cytokine-induced Src activation can be suppressed by moderate shear stress (5 dynes /cm 2) and the ER stress inhibitor. Next, to investigate the sub-cellular activation pattern of Src and FAK in response to inflammatory cytokines and mechanical loading, we used lipid raft-targeting (Lyn-FAK and Lyn-Src) and non-lipid raft-targeting (KRas-FAK and KRas-Src) biosensors. We also developed a three-dimensional (3D) cell culture system using collagen-coupled agarose gels to mimic the physiologically relevant cell microenvironment. The activities of Lyn-Src, KRas-Src and Lyn-FAK were up and down regulated by high (\u3e10 μl/min) and moderate (5 μl/min) interstitial fluid flow, respectively, but KRas-FAK did not respond to the flow. We also found that Src activation by loading was blocked by inhibition of FAK, while inhibition of Src did not affect FAK activities, suggesting that FAK is necessary for interstitial fluid flow-induced Src activity. In contrast, Src was necessary for inflammatory cytokine-induced FAK activation. Furthermore, we developed a 3D ex vivo system that uses murine cartilage explants. This system in conjunction with 3D FRET imaging allowed us to visualize sub-cellular signaling activities of Src and FAK that closely mimic in vivo setting. We found that intermediate loading can inhibit inflammatory cytokine-induced activities of Lyn-Src, KRas-Src and Lyn-FAK, but not KRas-FAK. AMP-activate kinase (AMPK) is a master regulator of cellular energy balance that activate when the ratio of (AMP+ADP)/ATP increases. Imbalance of AMPK regulation contributes to the development of diabetes, cardiovascular diseases, cancer, and found most recently, OA. In healthy cartilage, the treatment of IL1β and TNFα decreases AMPK activity, while how mechanical loading influences AMPK activities needs to be clarified. The recent design of FRET-based AMPK biosensors that can target various subcellular compartments (PM-AMPK targets plasma membrane, Cyto-AMPK target cytosol, Nuc-AMPK targets nucleus, ER-AMPK targets endoplasmic reticulum (ER), Golgi-AMPK targets Glogi apparatus, and Mito-AMPK targets mitochondria) enable the study about the regulation of AMPK compartmentalization by physical stimuli. The AMPK activities were upregulated by shear stress in 2D environment, while only Nuc- and PM-AMPK are responsive to loading in 3D environment, suggesting culture dimensionality alters the mechanosensitivity of chondrocytes. Moreover, to examine potential factors responsible for discrepancies between different culture models, we evaluated roles of cytoskeleton in mechanotransduction in 2D and 3D cultures and found that the differential cytoskeletal networks contribute to the different signaling in 2D versus 3D models

DIFFERENTIAL RHOA ACTIVITY IN CHONDROCYTES UNDER FLOW

poster abstractMechanical force environment is a major factor that influences cellular homeostasis and remodeling. The prevailing wisdom in this field demon-strated that a threshold of mechanical forces or deformation was required to affect cell signaling. However, we hypothesized that RhoA activities can be either elevated or reduced by selecting different levels of shear stress inten-sities. To test this hypothesis, a fluorescence resonance energy transfer (FRET)-based approach was used. The result revealed that C28/I2 chondro-cytes exhibited an increase in RhoA activities in response to high shear stress (10 or 20 dyn/cm2), while they showed a decrease in their RhoA activ-ities to intermediate shear stress at 5 dyn/cm2. No changes were observed under low shear stress (2 dyn/ cm2). The observed two-level switch of RhoA activities was closely linked to the shear stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. Collectively, these results here suggest that intensities of shear stress are critical in differential activa-tion and inhibition of RhoA activities in chondrocytes

eIF2α signaling regulates autophagy of osteoblasts and the development of osteoclasts in OVX mice

Bone loss in postmenopausal osteoporosis is induced chiefly by an imbalance of bone-forming osteoblasts and bone-resorbing osteoclasts. Salubrinal is a synthetic compound that inhibits de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Phosphorylation of eIF2α alleviates endoplasmic reticulum (ER) stress, which may activate autophagy. We hypothesized that eIF2α signaling regulates bone homeostasis by promoting autophagy in osteoblasts and inhibiting osteoclast development. To test the hypothesis, we employed salubrinal to elevate the phosphorylation of eIF2α in an ovariectomized (OVX) mouse model and cell cultures. In the OVX model, salubrinal prevented abnormal expansion of rough ER and decreased the number of acidic vesiculars. It regulated ER stress-associated signaling molecules such as Bip, p-eIF2α, ATF4 and CHOP, and promoted autophagy of osteoblasts via regulation of eIF2α, Atg7, LC3, and p62. Salubrinal markedly alleviated OVX-induced symptoms such as reduction of bone mineral density and bone volume fraction. In primary bone-marrow-derived cells, salubrinal increased the differentiation of osteoblasts, and decreased the formation of osteoclasts by inhibiting nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Live cell imaging and RNA interference demonstrated that suppression of osteoclastogenesis is in part mediated by Rac1 GTPase. Collectively, this study demonstrates that ER stress-autophagy axis plays an important role in OVX mice. Bone-forming osteoblasts are restored by maintaining phosphorylation of eIF2α, and bone-resorbing osteoclasts are regulated by inhibiting NFATc1 and Rac1 GTPase

Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells

Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs