Chelation of intracellular Ca²⁺ inhibits murine keratinocyte differentiation in vitro

Abstract

The role of intracellular Ca2+ in the regulation of Ca(2+)-induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca2+ 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 Ca2+ 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 microM) 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 loricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca2+, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca2+, the concentration of intracellular free Ca2+ (Cai) in BAPTA-loaded keratinocytes was examined by digital image analysis using the Ca(2+)-sensitive fluorescent probe fura-2, and Ca2+ influx was measured by 45Ca2+ uptake studies. Increase in extracellular Ca2+ (Cao) in the culture medium of keratinocytes caused a sustained increase in both Cai and Ca2+ localized to ionomycin-sensitive intracellular stores in keratinocytes. BAPTA lowered basal Cai concentration and prevented the Cai increase. After 12 hours of BAPTA treatment, the basal level of Cai returned to the control value, but the Ca2+ localized in intracellular stores was substantially decreased. 45Ca2+ uptake was initially (within 30 min) increased in BAPTA-loaded cells. However, the total 45Ca2+ accumulation over 24 hours in BAPTA-loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Cai and total cellular Ca2+ content in the presence of increased amount of intracellular Ca2+ buffer (e.g., BAPTA) by depleting intracellular Ca2+ 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 Ca2+ 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 Cai change.

Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis

“…S1P has been reported to induce Ca 2ϩ signaling, a key process for epidermal and keratinocyte differentiation (5,6,31). The regulated release of Ca 2ϩ from ER and Golgi stores, as well as the Ca 2ϩ influx through Ca 2ϩ -permeable ion channels, induces the transcription of genes associated with keratinocyte differentiation, such as keratin 1 and 10, filaggrin, and loricrin (61). S1P has long been known to induce elevations of intracellular Ca 2ϩ levels through the release from internal stores (5, 6), and its metabolism is believed to serve as a complementary Ca 2ϩ signaling pathway to promote keratinocyte differentiation (31).…”

Calcium-dependent Involucrin Expression Is Inversely Regulated by Protein Kinase C (PKC)α and PKCδ

“…Incubation of cultured keratinocytes with calcium increases differentiation and expression of differentiation-associated genes (1)(2)(3). Moreover, the presence in vivo of an epidermal calcium gradient, with increasing calcium levels in the more differentiated layers, suggests a role for calcium in regulating epidermal differentiation (2, 4 -6).…”

Requirement of an AP-1 Site in the Calcium Response Region of the Involucrin Promoter

“…The basal layer contains the actively proliferating cells. As the keratinocytes migrate from the basal layer outward, numerous differentiation markers, such as involucrin (INV) 1 (2), transglutaminase (3), loricrin (4), keratins K1 and K10 (5), and filaggrin (6), become apparent in the spinous and granular layers. The fully differentiated cells then form the cornified layer of the stratum corneum.…”