The Role of IGF-1 and TRPV4 in Regulation of [Ca2+]i and Actin Organization in ATDC5 Chondrocytes

Lam, Andy
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University of Delaware
Cartilage is a thin layer of tissue composed of mainly water, collagen, and proteoglycans that forms a sliding area so that diarthrodial joints can easily function. Chondrocytes are the cells of this tissue that produces and maintains the cartilage of matrix and the phenotype and function of these cells is regulated by the organization of actin cytoskeleton. Two dimensional culture studies have shown that Insulin-like Growth Factor-1 (IGF-1) increased cell stiffness through regulation of actin cytoskeleton, which suppressed the activation of TRPV4. TRPV4, a Ca2+ channel, is an important regulator of intracellular Ca2+ and is an anabolic factor in chondrocytes for matrix synthesis. I hypothesize that IGF-1 has differential effects on the actin cytoskeletal organization and TRPV4 channel activity in ATDC5 chondrocyte cells in 2D/3D culture environments. ATDC5 chondrocytes were cultured in sodium alginate beads and treated with 4 different treatments: control, IGF-1 treatment, Rac-1 Inhibitor treatment, IGF-1+Rac-1 inhibitor treatment (n=10) and [Ca2+]i levels were measured with PTI and FURA-2AM protocol. ATDC5 chondrocytes were also grown in 2D culture environment (collagen II coated coverslips) and in 3D (collagen/agar matrix), treated with 4 different treatments, stained with 488 Alexa Fluor Phalloidin to visualize actin cytoskeleton, and imaged with Zeiss 510 laser scanning microscope. Chondrocytes were then cultured in micromass cultures and treated with the four different treatments to see if there were any changes to levels of proteoglycan synthesis. IGF-1 treated chondrocytes in 3D tended to increase [Ca2+]i and exhibited a biphasic response, which was different from what 2D studies had predicted. IGF-1 treatment resulted in increased actin fiber formation in both 2D and 3D culture environments, while the effects of Rac-1 inhibition seemed dependent on the dimensions of culture environment. This suggests that IGF-1 increases actin fiber formation through RhoA in 2D culture and through Rac-1 activation in 3D cultures. IGF-1 treated micromass cultures exhibited similar levels of proteoglycan content as control treatments, but Rac-1 inhibition led to lowered proteoglycan content. The effects of IGF-1 are dependent on the dimensions of the culture environment. In 2D the effects of IGF-1 are dependent on Rac-1 activation, but in 3D IGF-1’s effects are dependent on RhoA activation.
Research Subject Categories::MEDICINE::Morphology, cell biology, pathology::Cell biology , Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology