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S. Mukherjee, A. Giddabasappa, W. Xiao, B. Xu, S. Chaney, D. A. Fox; Altered Regulation of Cell Cycle Progression and Exit Increases Retinal Progenitor Cell (RPC) Proliferation and Neuronal Differentiation During Gestational Lead Exposure (GLE). Invest. Ophthalmol. Vis. Sci. 2009;50(13):4003.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we showed that GLE increased and prolonged the proliferation of RPCs resulting in a significantly increased number of late-born retinal neurons (rods and bipolar cells), but not Müller glial cells [ARVO 2007]. Affymetrix microarray and RT-qPCR studies showed that GLE upregulated cyclin D1 and cyclin B1 as well as certain pro-neuronal bHLH and HD genes [ARVO 2008]. Our present goals were to determine the spatiotemporal mRNA and protein expression levels of cell cycle genes that directly and differentially regulate retinal cell cycle progression and exit during GLE.
C57BL/6 female mice were exposed to water or 55 ppm lead throughout gestation and until postnatal day (PN)10: equivalent to human gestation period. Control and GLE retinas from embryonic day (E)16.5 to PN10 were used for RT-qPCR, Westerns and confocal immunohistochemistry (IHC).
RT-qPCR analysis revealed the following in GLE retinas, compared to controls. Cyclin D1 was significantly upregulated at PN2 and PN6. p27[Kip1] was significantly downregulated at E16.5 and E18.5. Retinoblastoma1 [Rb1] was significantly downregulated at E16.5 and upregulated at PN2. Immunoblots of GLE retinas, compared to controls, showed a significant upregulation of cyclin D1 at PN4 and PN6; upregulation of p27 at PN2 and PN4; downregulation of Rb1 at PN6 and upregulation of phospho-Rb at PN2 to PN6; and downregulation of p16[INK4A] from PN2 to PN6. There were no changes in Cdk4 protein expression in GLE retinas relative to controls. IHC triple-labeling experiments revealed an increase in the number of cyclin D1-positive RPCs at PN3 and PN5, and of p27-positive RPCs from E16.5 to PN7 in GLE retinas relative to controls.
In developing GLE retinas, p16 [inhibitor of cyclin D1-Cdk4/6 activity] protein expression decreased while cyclin D1 and phospho-Rb protein expression increased. These changes are consistent with an increased G1-/S-phase transition as well as the increased and prolonged proliferation of RPCs observed in developing GLE retinas. The increased protein expression and number of p27-positive cells in GLE retinas coincides with the increased number of RPCs exiting cell cycle and differentiating into rods and/or bipolar cells. Overall, these results show that GLE selectively alters several direct regulatory steps in the G1-phase of cell cycle progression, which results in the unique adult retinal GLE phenotype.
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