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Emilia A Zin, Yan Zhang, Christine Frances Wildsoet; Identification of Elements of the BMP2 Signaling Pathway in Cultured Chick Scleral Fibroblast. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2172.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have indicated the role of Bone Morphogenetic Protein 2 (BMP2) in eye growth regulation. This study investigated the role of BMP2 in scleral remodeling using a chick scleral fibroblast culture model to look for evidence of BMP2 signaling.
Primary chick scleral fibroblast (CSF) were cultured on 24-well plates or chamber slides in DMEM/F-12 medium with 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. CSF total RNA was collected and purified using RNeasy Mini Kit and then subjected to cDNA synthesis and real-time PCR semi-quantification. Gene expression was examined for BMP2, BMP receptors (BMPR1A, 1B, -2), SMAD1, -5, and -9, and the localization of relevant proteins, BMP2, BMPR1A, BMPR2, SMAD1, -4, and -5, phosphorylated SMAD1 (p-SMAD1), and p-SMAD1/5 in CSF and 293T cell lines was investigated using immunocytochemistry. Protein expression was also validated with Simon automated western blot.
Cultured CSF showed detectible expression of BMP2, BMP receptors, and SMAD 1, 5, 9 genes and immunohistochemistry confirmed the expression of BMPR1A, BMPR2, SMAD1, -4, and -5 proteins in CSF as well as 293T cells. Western blot analyses confirmed expression of the p-SMAD1/5 protein in both CSF and 293T cells, while SMAD1 and p-SMAD1 proteins were only detected in 293T cells.
That cultured chick scleral fibroblasts express many of the components of the BMP2 signaling pathway, at both gene and protein levels, points to their likely involvement in scleral remodeling and ocular growth. These results provide a foundation for future in vivo studies into the role of BMP2 in ocular (scleral) growth regulation.
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