Abstract
Purpose::
The purpose of this paper was to characterize mouse inner medullary collecting duct cell line (mIMCD3) and retinal astrocyte cell line (WTRAC) for Pax2 regulation studies by Bone Morphogenetic Protein7 and Sonic Hedgehog. Pax2 is a member of paired box family of homeotic genes and comes under Pax2/5/8 subfamily. It is a nuclear transcription factor that plays an important role in the development of eye, ear, kidney, uterus, pancreas and spinal cord. Pax2 is essential for fusion of optic fissure, differentiation of epithelial cells in urogenital tract, patterning of hindbrain and inner ear.It is also expressed in astrocytes that migrate into the eye in mammals.
Methods::
Characterization of mIMCD3 and WTRAC cells was done by immunocytochemical analysis for Pax2, Pax6, Patched, phospho smad1, smad5, Vimentin and GFAP(done in WTRAC cells). Cells were seeded at a concentration of 2 X 105 cells/dish and then treated with 100ng/ml BMP7, 200ng/ml shh and a combination of both. Total RNA was isolated from control and treated cells and analyzed by RT-PCR for Pax2. Western analysis for Pax2, P-Pax2, Smad-1, P-smad-1, smad-2 and P-JNK was done on protein isolated from vehicle and treated WTRAC and mIMCD3 cells.
Results::
1.Both mIMCD3 and WTRAC cells are positive for Pax2, Ptc, BMP7, smad-5 and P-smad 1, which makes them a good system for our studies.2.mIMCD3 cells showed an increase in Pax2 and P-Pax2 protein levels on treatment with BMP7 and shh. A further increase in Pax2 and P-Pax2 was observed when the cells were treated with a combination of BMP7 and shh.3.Shh increased the levels of P- smad1 in mIMCD3 cells, which indicated a possible link between BMP7 and shh pathways.4.WTRAC cells also showed an increase in Pax2 and P-Pax2 protein levels after treatment with BMP7.5. RT-PCR results showed an increase in Pax2 mRNA levels in mIMCD3 cells, after treatment with shh as well as shh in combination with BMP7.6.In WTRAC cells there was no detectable Pax2 mRNA in control and BMP7 treated cells. In shh treated cells Pax2 mRNA was detectable which increased in the cells treated with BMP7 and shh together.
Conclusions::
1. Activation of P-Smad1 by shh in mIMCD3 indicates a crosstalk between BMP7 and shh pathways.2.No detectable mRNA in control and BMP7 treated WTRAC cells and increase in Pax2 mRNA levels after treatment with BMP7 and shh in combination, indicates that BMP7 and shh together might be contributing to the stablity of Pax2 mRNA.
Keywords: astrocytes: optic nerve head • optic nerve • astrocyte