Effects of extracellular calcium on the growth‐differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes

Abstract

Abstract:The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0 ⁄ G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro, the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 mm. In NHK, addition of calciuminduced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S ⁄ G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.

Role of STIM1- and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation

“…Expression of KRT1 occurs mainly in the suprabasal layer and stratum spinosum in human epidermis (Yuspa et al, 1988). It has been reported that HaCaT cells differentiate in response to elevated Ca 2+ in a manner similar to normal epidermal keratinocytes, but HaCaT cells require somewhat higher extracellular Ca 2+ concentration and a longer time to launch the differentiation process (Micallef et al, 2009;Sakaguchi et al, 2003). As shown in Fig.…”

“…cDNA synthesis was performed with Omniscript (Qiagen), using 0.5 mg total RNA, and quantitative RT-PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems) with the ABI Prism 7000 Instrument (Applied Biosystems), using specific oligonucleotides [human KRT1, forward 59-ATTTCTGAGCTGAATCGTGTGATC-39, reverse 59-CTTGGCATC-CTTGAGGGCATT-39 (Micallef et al, 2009); GAPDH, forward 59-GAAGG-TGAAGGTCGGAGTC-39, reverse 59-GAAGATGGTGATGGGATTTC-39].…”

“…S2). Induction of KC differentiation is associated with proliferation arrest (Fuchs, 1993;Micallef et al, 2009). We and others have reported that STIM1 and Orai1 have critical roles in proliferation and cell cycle regulation (Abdullaev et al, 2008;Chen et al, 2011;Smyth et al, 2009).…”

Cadherin-based intercellular adhesions organize epithelial cell–matrix traction forces

“…Because elevation of extracellular calcium modulates cellular properties in addition to cadherin-based-adhesion induction (43,44), we sought to isolate the role of cadherin in spatially organizing traction forces. We used two different methods to inhibit formation of cadherinbased adhesions in the presence of high-calcium medium.…”

TRP Channels and Pruritus

“…Calcium signaling is a key element of keratinocyte differentiation [357][358][359][360] and, as we suggested above, altered differentiation of epidermal keratinocytes may play a role in pruritic diseases such as e.g. AD and psoriasis [361].…”