The regulation of calcitonin genes upon bacterial infection and sepsis in human adipocytes

Systemic bacterial infections induce an ubiquitous expression of calcitonin (CALC) genes with sustained release of calcitonin (CT) peptides, namely procalcitonin (ProCT), calcitonin-gene related peptide (CGRP) and adrenomedullin (ADM). ProCT is a marker to follow the course of sepsis and to guide antibiotic therapy and a dose dependent toxic mediator. Persistently and markedly elevated levels of ProCT and even more of ProADM during bacterial infection and sepsis indicate a bad prognosis. The molecular mechanisms as to how extrathyroidal CALC gene expression and protein secretion is regulated in sepsis are unknown.
Since the neuro-endocrine CT expression in the parafollicular C-cells of the thyroid is calcium dependent, we hypothesized that calcium might also be involved in the nonneuro-endocrine expression and secretion of CT peptides. We therefore monitored preadipocyte-derived adipocytes for changes in intracellular calcium concentrations upon treatment with lipopolysaccharide (LPS) with confocal microscopy. LPS stimulated cells were treated with substances which provoke or block an increase in intracellular calcium concentrations or increase levels of cAMP and changes in CALC-I gene (i.e. CT and CGRP-I) and CALC-V gene (i.e. ADM) mRNA expression were assessed by real-time PCR. Protein secretions in supernatants were measured by specific assays. In addition to the CALC genes, changes of IL-6 mRNA and protein were measured.
Administration of LPS on human adipocytes led to a slow and sustained increase in the intracellular calcium concentration without apparent cytotoxic effect on the cells. LPS-induced CALC-I mRNA expression was potentiated with increasing intracellular calcium concentrations through addition of the calcium ionophore ionomycin or depletion of intracellular stores with thapsigargin. When diminishing intracellular calcium concentrations in LPS-treated adipocytes by verapamil or 2- aminoethoxydiphenyl borate, the CALC-I gene expression was reduced. This was confirmed at the protein level for ProCT and CGRP-I.
Interestingly, we observed an inverse effect of intracellular calcium on the expression of the CALC-I and the CALC-V gene. Elevations of intracellular calcium in an inflammatory background caused by LPS potentiated the activity of the CALC-I genes CT and CGRP-I but reduced the expression of the CALC-V gene ADM, both at the mRNA and the protein level. IL-6 mRNA expression levels was also altered upon increased and decreased levels of intracellular calcium concentrations, but to a much lower extend as compared to that of the CALC-I gene. LPS-induced expression of CT and CGRP-I mRNA was independent from nuclear factor kappa B (NFκB), in contrast to IL-6-expression.
In conclusion, the expression of CT and CGRP-I in human preadipocyte-derived adipocytes upon stimulation with LPS is mediated by changes in intracellular calcium concentrations and by cAMP and is independent of NFκB. The distinct and inverse effect of calcium on CALC-I and CALC-V gene expression might at least in part explain the different clinical characteristics of ProCT as diagnostic and ProADM as prognostic marker.