Expression of the CT/CGRP gene and its regulation by dibutyryl cyclic adenosine monophosphate in human osteoblastic cells

There is general agreement that calcitonin (CIT) inhibits bone resorption by its effects on osteoclast function, CT was also found to have direct effects on osteoblast-like cells, In this study, we investigated the expression of CT and calcitonin gene-related peptide (CGRP), the two peptides encoded by the CT/CGRP gene, in human osteosarcoma cell lines and in normal human trabecular osteoblastic cells (HOB), and we studied the modulation of CT/CGRP gene expression by dibutyryl cyclic adenosine monophosphate ((Bu)(2) cAMP), a cAMP analog, We first detected by Northern blot hybridization the presence of CT and CORP mRNAs in different osteosarcoma cell lines (OHS-4, MG-63, Saos-2, HOS-TE85) and HOB cells, In the steady state, OHS-4 cells express slightly more CT and CGRP mRNAs than other cell lines or normal human osteoblasts, in parallel with messengers of differentiated osteoblasts, such as osteocalcin (OC) and alkaline phosphatase (ALP), OHS-4 cells also express CT and CGRP proteins, as demonstrated by immunocytochemistry, Stimulation of OHS-4 cells with 1 mM (Bu)(2) cAMP induced a significant increase in mRNA levels for CT (x2.5) and CGRP (x3), as determined by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure, The involvement of a transcriptional mechanism in this effect was evidenced by nuclear run-off transcription assay, In addition, (Bu)(2) cAMP increased OC (x4) and ALP (x3) mRNA levels in OHS-4 cells, These effects were observed at 24 h and were maximal at 48 h, indicating that (Bu), cAMP induced cell differentiation and increased the transcription of the CT/CGRP gene in OHS-4 osteoblast-like cells, The results indicate that human osteosarcoma cells and primary human osteoblastic cells express CT and CGRP mRNA and proteins, and that (Bu)(2) cAMP, an activator of protein kinase A, induces up-regulation of osteoblastic phenotypic genes and enhances CT and CGRP gene transcription, indicating that induction of osteoblastic differentiation by (Bu)(2) cAMP is associated with enhanced expression of CT and CORP in human osteoblastic cells.