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.


OBJECTIVE
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.
Pleguezuelos
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Molecular Signalling Group, Clinical and Diagnostic Oral Sciences, Barts & The London, Queen Mary University of London, 4 Newark Street, London E1 2AT, UK.
Kapas
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D053607:Adrenomedullin; D000328:Adult; D002122:Calcium Chloride / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D015850:Interleukin-6; D016328:NF-kappa B; D010455:Peptides; D053607:Adrenomedullin; D002122:Calcium Chloride