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Chelation of intracellular Ca 2+ inhibits murine keratinocyte differentiation in vitro
Author(s) -
Li Luowei,
Tucker Robert W.,
Hennings Henry,
Yuspa Stuart H.
Publication year - 1995
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041630112
Subject(s) - in vitro , chelation , intracellular , microbiology and biotechnology , chemistry , keratinocyte , pharmacology , biology , biochemistry , organic chemistry
The role of intracellular Ca 2+ in the regulation of Ca 2+ ‐induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca 2+ chelator 1,2‐bis(o‐aminophenoxy)‐ethane‐N, N, N′, N′‐tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca 2+ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca 2+ . Intracellular BAPTA loaded by BAPTA/AM (15–30 μM) inhibited the expression of epidermal differentiation‐specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation‐associated redistribution of E‐cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and Ioricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca 2+ , indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca 2+ , the concentration of intracellular free Ca 2+ (Ca i ) in BAPTA‐loaded keratinocytes was examined by digital image analysis using the Ca 2+ ‐sensitive fluorescent probe fura‐2, and Ca 2+ influx was measured by 45 Ca 2+ uptake studies. Increase in extracellular Ca 2+ (Ca o ) in the culture medium of keratinocytes caused a sustained increase in both Ca i and Ca 2+ localized to ionomycin‐sensitive intracellular stores in keratinocytes. BAPTA lowered basal Ca i concentration and prevented the Ca i increase. After 12 hours of BAPTA treatment, the basal level of Ca i returned to the control value, but the Ca 2+ localized in intracellular stores was substantially decreased. 45 Ca 2+ uptake was initially (within 30 min) increased in BAPTA‐loaded cells. However, the total 45 Ca 2+ accumulation over 24 hours in BAPTA‐loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Ca i and total cellular Ca 2+ content in the presence of increased amount of intracellular Ca 2+ buffer (e.g., BAPTA) by depleting intracellular Ca 2+ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca 2+ ‐stores since this is the major change in intracellular Ca 2+ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E‐cadherin on the cell membrane may be more directly associated with Ca i change. © 1995 Wiley‐Liss, Inc.