Journal of Cell Science
Role of STIM1- and Orai1-mediated Ca2+entry in Ca2+-
induced epidermal keratinocyte differentiation
Takuro Numaga-Tomita and James W. Putney*
National Institute of Environmental Health Sciences – NIH, Department of Health and Human Services, PO Box 12233, Research Triangle Park,
NC 27709, USA
*Author for correspondence (email@example.com)
Accepted 11 November 2012
Journal of Cell Science 126, 605–612
? 2013. Published by The Company of Biologists Ltd
The uppermost thin layer on the surface of the skin, called the epidermis, is responsible for the barrier function of the skin. The
epidermis has a multilayered structure in which each layer consists of keratinocytes (KCs) of different differentiation status. The
integrity of KC differentiation is crucial for the function of skin and its loss causes or is accompanied by skin diseases. Intracellular and
extracellular Ca2+is known to play important roles in KC differentiation. However, the molecular mechanisms underlying Ca2+
regulation of KC differentiation are still largely unknown. Store-operated Ca2+entry (SOCE) is a major Ca2+influx pathway in most
non-excitable cells. SOCE is evoked in response to a fall in Ca2+concentration in the endoplasmic reticulum. Two proteins have been
identified as essential components of SOCE: STIM1, a Ca2+sensor in the ER, and Orai1, a subunit of Ca2+channels in the plasma
membrane. In this study, we analyzed the contribution of SOCE to KC growth and differentiation using RNAi knockdown of STIM1 and
Orai1 in the human keratinocyte cell line, HaCaT. KC differentiation was induced by a switch in extracellular Ca2+concentration from
low (0.03 mM; undifferentiated KCs) to high (1.8 mM; differentiated KCs). This Ca2+switch triggers phospholipase-C-mediated
intracellular Ca2+signals (Ca2+-switch-induced Ca2+response), which would probably involve the activation of SOCE. Knockdown of
either STIM1 or Orai1 strongly suppressed SOCE and almost completely abolished the Ca2+-switch-induced Ca2+responses, resulting in
impaired expression of keratin1, an early KC differentiation marker. Furthermore, loss of either STIM1 or Orai1 suppressed normal
growth of HaCaT cells in low Ca2+and inhibited the growth arrest in response to a Ca2+switch. These results demonstrate that SOCE
plays multiple crucial roles in KC differentiation and function.
Key words: Store-operated calcium entry, Calcium channels, Orai channels, Epidermal keratinocyte
The skin functions as physical protection for the body from
harmful pathogens, hazardous chemicals or dehydration. This
barrier function of the skin is carried out by the outermost thin
layer called epidermis. The epidermis is a multilayered structure
composed primarily of cells termed keratinocytes. Each layer of
the epidermis consists of keratinocytes in different differentiation
status. While undifferentiated keratinocytes in the basal layer are
highly proliferative, they exit the cell cycle and undergo
differentiation upon migrating to upper layers. Therefore, the
integrity of differentiation process of keratinocytes is critical for
skin barrier function. Defects in this barrier function are known to
cause many skin diseases (Proksch et al., 2008).
Many factors interact to induce KC differentiation including
calcium (Ca2+), vitamin D, and cell to cell contact (Bikle, 2010;
Dotto, 1999; Yuspa et al., 1988). It has been reported that there is
a gradient of extracellular Ca2+concentration in the epidermis
from low in the basal layer to high in the stratum granulosum, the
uppermost layer of epidermis (Pillai et al., 1993). Indeed,
increases of extracellular Ca2+concentration (Ca2+switch) can
induce KCs to exit the cell cycle and express several
differentiation markers in isolated primary KCs in vitro (Pillai
et al., 1990). Furthermore, low extracellular Ca2+concentration
is critical to maintain the highly proliferative nature of
undifferentiated KCs. It has previously been shown that the
Ca2+switch is sensed by a Ca2+-sensing receptor (CaR) in the
plasma membrane of KCs (Tu et al., 2004). CaR is a G-protein-
coupled receptor coupled to Gqtype alpha subunits, and thus
activation of CaR leads to activation of the phospholipase C
pathway (Hofer and Brown, 2003). CaR-mediated PLC signaling
is initially mediated by PLCb and subsequently by PLCc (Xie
and Bikle, 1999). Suppression of the intracellular Ca2+increase
with chelators, or suppression of PLCc activity attenuate KC
differentiation, suggesting that Ca2+signaling is a key signaling
pathway for Ca2+-switch-induced KC differentiation (Li et al.,
1995). However, the exact molecular mechanism underlying
Ca2+-switch-induced Ca2+mobilization is largely unknown.
Several Ca2+-permeable channels are suggested to be involved
in Ca2+signaling in Ca2+-switch-induced KC differentiation
including transient receptor potential family channels (Beck et al.,
2008; Cai et al., 2006; Mu ¨ller et al., 2008).
Store-operated Ca2+entry (SOCE) is a major Ca2+influx
pathway in most non-excitable cells (Parekh and Putney, 2005).
As its name suggests, SOCE is activated by depletion of Ca2+
stores in the endoplasmic reticulum (ER). SOCE is known to be
involved in cell proliferation and differentiation processes
(Darbellay et al., 2009; Hwang and Putney, 2012; Johnstone
et al., 2010). SOCE is mediated essentially by two classes of
proteins, the STIM and Orai proteins (Feske et al., 2006;
Liou et al., 2005; Roos et al., 2005; Vig et al., 2006; Zhang
Journal of Cell Science
values at the wavelength of 475 nm and 660 nm were measured using a microtiter
plate reader. Average of blank-subtracted absorbance was calculated from
triplicate readings and normalized to that of siControl-transfected cells cultured
72 hours in 0.03 mM Ca2+-containing KGM-2.
For quantitative RT-PCR, total RNAs were extracted from HaCaT cells with the
RNeasy mini kit (Qiagen). 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].
This research was supported by the intramural program, National
Institutes of Health [Project #01 ES090087]. Deposited in PMC for
release after 12 months.
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