Bone Abstracts

Objective: The physis, or growth plate, is comprised of precisely organized chondrocytes that confer longitudinal growth of the bone. Multiple signaling pathways cooperate to regulate growth through their control of chondrocyte shape, polarity, proliferation, and differentiation.^1,2 Disruption of these cellular events result in physeal defects, skeletal deformities, and abnormal limb growth. Loss of function mutations in Smad4, a common intracellular effector of all transforming growth factor β (TGFβ) family members, is responsible for human Myhre syndrome characterized by short stature, brachydactyly, and joint stiffness.³ In the physis of Smad4 deficient mice, the columnar organization is replaced with a disorganized mass of misshapen chondrocytes.⁴ Although many studies affirm the key role for Smad4/TGFβ family signaling in chondrocytes, the mechanisms by which Smad4 maintains normal growth plate organization are not defined.

Methods: Tibias of Col2a1-Cre;Smad4^fl/fl mice were isolated at e18.5 and frozen sections were used for immunofluorescence and immunohistochemistry. Primary chondrocytes were isolated from Smad4^fl/fl mice, underwent adenoviral-cre infection and cultured in a pellet for 21 days.

Results: Our data indicate Smad4 is responsible for maintaining normal proliferation, columnar organization and proteoglycan synthesis in vivo and in vitro with consistent results in the physis and primary murine chondrocyte pellet cultures. Using immunofluorescence, we determined several markers of cell polarity, including cell shape and direction of cell elongation, are aberrant in Smad4-deficient chondrocytes. The number of cells in a proliferative column and markers of proliferation are reduced. Proteoglycan content, through Safranin-O staining and immunohistochemistry, is also reduced.

Conclusions: Smad4 is involved in cytoskeleton reorganization and proteoglycan synthesis in the physis and could alter the extracellular signaling of many critical pathways, including the planar cell polarity (PCP) pathway. This study enriches our knowledge in the development and regulation of the growth plate, cartilage biology, tissue engineering, joint formation, articular cartilage development, and osteoarthritis.

References: 1. Abad V et al. Endocrinology 1999 140 (2) 958–962.

2. Sasaki A et al. J Biol Chem 2001 276 (21) 17871–17877.

3. Caputo V et al. Am J Hum Genet 2012 90 (1) 161–169.

4. Zhang J et al. Dev Biol 2005 284 (2) 311–322.