S J Millward-Sadler

Mechanotransduction via integrins and interleukin-4 results in altered aggrecan and matrix metalloproteinase 3 gene expression in normal, but not osteoarthritic, human articular chondrocytes

OBJECTIVE To determine molecular events in the regulation of messenger RNA (mRNA) of cartilage matrix molecules and proteases by mechanical stimulation of chondrocytes from normal human articular cartilage and to ascertain whether similar regulatory systems are present in chondrocytes from osteoarthritic (OA) cartilage. METHODS Chondrocytes extracted from macroscopically and microscopically normal and OA cartilage were mechanically stimulated in the presence or absence of GRGDSP or GRADSP oligopeptides, neutralizing interleukin-4 (IL-4) antibodies, gadolinium, or apamin. The relative levels of mRNA for aggrecan, tenascin, matrix metalloproteinase 1 (MMP-1), MMP-3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) were determined by semiquantitative reverse transcription-polymerase chain reaction at several time points up to 24 hours poststimulation, using GAPDH as a control. RESULTS Normal chondrocytes showed an increase in aggrecan mRNA and a decrease in MMP-3 mRNA within 1 hour following stimulation, with a return to baseline levels within 24 hours. These changes were blocked by GRGDSP, IL-4 antibodies, and gadolinium, but were unaffected by apamin. In contrast, chondrocytes isolated from OA cartilage showed no change in aggrecan or MMP-3 mRNA levels following mechanical stimulation. The mRNA levels of tenascin, MMP-1, and TIMP-1 were unaltered in mechanically stimulated normal and OA chondrocytes. CONCLUSION Mechanical stimulation of human articular chondrocytes in vitro results in increased levels of aggrecan mRNA and decreased levels of MMP-3 mRNA. The transduction process involves integrins, stretch-activated ion channels, and IL-4. This chondroprotective response is absent in chondrocytes from OA cartilage. Abnormalities of mechanotransduction leading to aberrant chondrocyte activity in diseased articular cartilage may be important in the progression of OA.

Integrin-dependent signal cascades in chondrocyte mechanotransduction

Mechanical forces influence chondrocyte metabolism and are critically important for maintenance of normal cartilage structure and integrity. In cells of the musculoskeletal system and mechanoresponsive cells in other tissues, integrins seem to be involved in the mechanotransduction process. Integrin activity is important in the early cellular responses to mechanical stimulation, regulating activation of a number of intracellular cascades that induce changes in gene expression and tissue remodeling. In normal human articular chondrocytes, integrin activation, consequent to mechanical stimulation in vitro, results in tyrosine phosphorylation of regulatory proteins and subsequent secretion of autocrine and paracrine acting soluble mediators including substance P and interleukin 4. Significant differences in signaling events and cellular responses are seen when normal and osteoarthritic chondrocytes are mechanically stimulated. These differences may relate to differences in integrin expression and function. Improved comprehension of how integrins mediate chondrocyte responses to mechanical stimulation, and how cross talk between integrin signaling, extracellular matrix, and autocrine/paracrine signaling molecules regulate mechanotransduction and cellular reactions are necessary for further understanding of how load influences cartilage structure.

Integrin and Mechanosensitive Ion Channel-Dependent Tyrosine Phosphorylation of Focal Adhesion Proteins and β-Catenin in Human Articular Chondrocytes After Mechanical Stimulation

Mechanical forces influence chondrocyte metabolism and function. We have previously shown that 0.33 Hz cyclical pressure‐induced strain (PIS) results in membrane hyperpolarization of normal human articular chondrocytes (HAC) by activation of Ca2+‐dependent K+ small conductance potassium activated calcium (SK) channels. The mechanotransduction pathway involves α5β1‐integrin, stretch‐activated ion channels (SAC) actin cytoskeleton and tyrosine protein kinases, with subsequent release of the chondroprotective cytokine interleukin‐4 (IL‐4). The objective of this study was to examine in detail tyrosine phosphorylation events in the mechanotransduction pathway. The results show tyrosine phosphorylation of three major proteins, p125, p90, and p70 within 1 minute of onset of mechanical stimulation. Immunoblotting and immunoprecipitation show these to be focal adhesion kinase (pp125FAK), β‐catenin, and paxillin, respectively. Tyrosine phosphorylation of all three proteins is inhibited by RGD containing oligopeptides and gadolinium, which is known to block SAC. β‐catenin coimmunoprecipitates with FAK and is colocalized with α5‐integrin and pp125FAK. These results indicate a previously unrecognized role for an integrin‐β‐catenin signaling pathway in human articular chondrocyte (HAC) responses to mechanical stimulation.

Integrin-interleukin-4 mechanotransduction pathways in human chondrocytes.

Mechanical stimuli are known to have major influences on chondrocyte function. The molecular events that regulate chondrocyte responses to mechanical stimulation are beginning to be understood. In vitro analyses have allowed identification of mechanotransduction pathways that control molecular and biochemical responses of human articular chondrocytes to cyclical mechanical stimulation. These studies have shown that human articular chondrocytes use alpha5beta1 integrin as a mechanoreceptor. After stimulation of this integrin by mechanical stimulation, there is activation of a signal cascade, involving stretch-activated ion channels, the actin cytoskeleton and tyrosine phosphorylation of the focal adhesion complex molecules pp125 focal adhesion kinase and paxillin, and beta-catenin. Subsequently, there is secretion of interleukin-4, which acts in an autocrine manner via Type II receptors, to induce membrane hyperpolarization, increase levels of aggrecan messenger ribonucleic acid, and decrease levels of matrix metalloproteinase 3 messenger ribonucleic acid. Chondrocytes from osteoarthritic cartilage also use alpha5beta1 integrin as a mechanoreceptor, but downstream signaling cascades and cell responses including changes in aggrecan messenger ribonucleic acid are different. Abnormalities of chondroprotective mechanotransduction pathways in osteoarthritis may contribute to disease progression.