Subphysiological concentrations of extracellular calcium sensitize normal human keratinocytes to UVB-induced apoptosis

Abstract

Normal human keratinocytes are stimulated to proliferate in serum-free medium containing subphysiological concentrations of calcium (0.09 mM, low calcium). In this study, we examined the effect of increased levels of extracellular calcium (2.0 mM, normal calcium) on UVB-induced apoptosis. Apoptosis was assessed by changes in cellular morphology, annexind V-FITC flow cytometry, and the formation of internucleosomal DNA ladders. High doses of UVB induced keratinocytes grown in low calcium medium to undergo apoptosis. In contrast, keratinocytes grown for 72 h in normal calcium medium were completely resistant to UVB-induced apoptosis. No apoptosis was observed even at UVB doses as high as 1200 J/m2. However, despite the lack of UVB-induced cell death, keratinocytes grown in normal calcium medium lost the ability to proliferate following high levels of UVB irradiation. High doses of UVB also increased the expression of the differentiation-specific proteins involucrin and cytokeratin 10 in a dose-dependent manner. In addition, growth in normal calcium medium lowered the UVB-induced stimulation of the p53 protein and altered the normal subcellular localization pattern of p53. UVB irradiation of human keratinocytes grown in normal calcium medium may be inducing further cell differentiation in the absence of overt cell death.

Induction of matrix metalloproteinase-9 secretion from human keratinocytes in culture by ultraviolet B irradiation

“…The expression of involucrin, a marker of keratinocyte differentiation, has been reported to be induced after UVB irradiation using human keratinocytes in culture [35]. Furthermore, UVB irradiation up-regulates involucrin expression in vivo [31,36].…”

Characterization of a Human Skin Equivalent Model to Study the Effects of Ultraviolet B Radiation on Keratinocytes

“…65 This has been attributed to factors such as keratinocyte differentiation status and cellular adhesion. 66 The UVB-triggered secretion of inflammatory cytokines, including IL-1, -6, -8, -10, -12, and tumor necrosis factor-a, stimulates erythemal and immunomodulatory responses postirradiation. [67][68][69][70] In line with earlier reports, our data demonstrate a significant augmentation of IL-6 and IL-8 levels in HSE-DED culture media after UVB irradiation.…”

Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death

“…UVR, calcium levels, and presence or absence of growth factors have all been shown to affect expression of differentiation markers in human keratinocytes as well as to affect apoptosis. 23,25,26 To evaluate whether the increased susceptibility to apoptosis in the filaggrin-expressing REK lines was due to the inducible expression of human filaggrin rather than induction of endogenous rat filaggrin, we evaluated the cell lines for expression of rat filaggrin after apoptotic stimuli by immunoblotting. Figure 6 demonstrates that there is no increase in either the tetracycline-inducible human filaggrin or endogenous rat filaggrin after various apoptotic stimuli.…”