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Keywords:

  • parathyroid gland;
  • calcium-sensing receptor;
  • glial cells missing;
  • vitamin D receptor;
  • transcription factor

Abstract

Glial cells missing-2 (Gcm2) is the key regulating transcription factor for parathyroid gland development. The continued expression of high levels of Gcm2 in mature parathyroid glands suggests that it is required for maintenance of parathyroid cell differentiation. The role of Gcm2 in parathyroid cell physiology, however, has not been fully studied. In this study, we examined the effects of Gcm2 silencing on cultured human parathyroid cells. Collagenase-dispersed human parathyroid cells from patients with chronic kidney disease were placed in monolayer cultures and infected with lentivirus expressing shRNA for human Gcm2. Seventy-two hours after infection, mRNA was processed and analyzed for Gcm2, PTH, vitamin D receptor (VDR), calcium-sensing receptor (CaR), 25-hydroxyvitamin D3 1-α-hydroxylase (1-OHase), and proliferating cell nuclear antigen (PCNA) by real-time PCR (qPCR). Protein expression of affected genes was analyzed by immunoblot 72 h after infection. Gcm2 mRNA and protein were decreased by 74.2 ± 12.2% (SD; n = 3 experiments; p < 0.01) and 67.5 ± 15.7% (n = 2; p < 0.01), respectively. CaR mRNA and protein were reduced by 47.8 ± 21.1% (n = 3; p < 0.01) and 48.1 ± 4.3% (n = 3; p < 0.01), respectively. However, VDR, PTH, 1-OHase, and PCNA were not significantly affected by Gcm2 silencing. Further analysis of CaR mRNA indicated that transcripts containing exon 1B, derived by transcription from CaR promoter 2, were downregulated (58.8 ± 19.27%; n = 3; p < 0.05) by Gcm2 silencing. Exon 1A–containing transcripts from promoter 1 were expressed at very low levels in the cultures. These results indicate that one function of Gcm2 is to maintain high levels of CaR expression in parathyroid cells.