Differentiation of the HaCaT keratinocyte cell line: modulation by adrenomedullin.

Background: Adrenomedullin (AM) is a multifunctional peptide produced by a wide variety of cells, including keratinocytes. We, and others, have demonstrated that AM has a role as a growth regulatory factor of the skin and contributes as an antimicrobial agent in the integument's protective barrier. It is not known whether AM has a role in differentiating keratinocytes.

Objectives: To study the role of AM in keratinocyte differentiation, modulating the effects of calcium and in addition, to assess whether differentiated keratinocytes are still capable of initiating an inflammatory response.

Methods: HaCaT cells were differentiated using CaCl2. Expression of transglutaminase type 1 (TG1) and E2F1 genes was used to monitor differentiation. AM secretion was measured by enzyme-linked immunosorbent assay (ELISA). NF-kappaB activity and interleukin (IL)-6 secretion in the cells were assessed after exposure to calcium and AM by electrophoretic mobility shift assay and ELISA, respectively.

Results: Secretion of AM by the keratinocyte cell line HaCaT was found to be increased during 1 mmol L(-1) CaCl2-induced cell differentiation but not 0.1 mmol L(-1) CaCl2. All treatments showed low levels of the cell proliferation marker, E2F1. Over time, cells incubated in the presence of 0.1 mmol L(-1) or 1 mmol L(-1) of CaCl2 showed an increase in TG1 expression, a marker of early differentiation. The addition of AM showed a decrease in TG1 expression when combined with 0.1 mmol L(-1) CaCl2, but not with 1 mmol L(-1) CaCl2. In addition, cells kept in 0.1 mmol L(-1) CaCl2 showed translocation of NF-kappaB after 48 h and 72 h of incubation, which was abolished when AM was added to the cells. Treatment with 1 mmol L(-1) CaCl2 led to earlier translocation of NF-kappaB at 24 h after treatment and addition of AM did not abolish the effect of 1 mmol L(-1) CaCl2 on NF-kappaB activation. Cells incubated in 0.1 mmol L(-1) CaCl2 showed increased secretion of IL-6 over time, consistent with NF-kappaB activation. The addition of AM to cells incubated with 0.1 mmol L(-1) CaCl2 showed a rapid decrease in IL-6 secretion after only 6 h. However, 1 mmol L(-1) CaCl2 did not induce secretion of IL-6 and the addition of AM did not affect the result.

Conclusions: Our data indicate that AM can reverse calcium-induced differentiation when 0.1 mmol L(-1) CaCl2 is used but not 1 mmol L(-1) CaCl2. Cells differentiated with 0.1 mmol L(-1) CaCl2 are still capable of generating an inflammatory response, showing signs of late NF-kappaB activation and IL-6 secretion that can be inhibited by AM. However, cells differentiated with 1 mmol L(-1) CaCl2 lose their ability to secrete IL-6 but not AM, which could be acting as an antimicrobial peptide.