Reversal of murine epidermal atrophy by topical modulation of calcium signaling.

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

Epidermal growth factor receptor down-regulation triggers human myoblast differentiation.

Initiation of human myoblast differentiation requires a negative shift (hyperpolarization) of the resting potential of myoblasts that depends on the activation of Kir2.1 potassium channels. These channels are regulated by a tyrosine phosphorylation. Using human primary myoblast culture, we investigated a possible role of various receptor tyrosine kinases in the induction of the differentiation process. We found that Epidermal Growth Factor Receptor (EGFR) is a key regulator of myoblast differentiation. EGFR activity is down-regulated during early human myoblast differentiation, and this event is required for normal differentiation to take place. Furthermore, EGFR silencing in proliferation conditions was able to trigger the differentiation program. This occurs through an increase of Kir2.1 channel activity that, via a rise of store-operated Ca(2+) entry, leads to the expression of myogenic transcription factors and muscle specific proteins (Myogenin, Myocyte Enhancer Factor 2 (MEF2), Myosin Heavy Chain (MyHC)). Finally, blocking myoblast cell cycle in proliferation conditions using a cdk4 inhibitor greatly decreased myoblast proliferation but was not able, on its own, to promote myoblast differentiation. Taken together, these results show that EGFR down-regulation is an early event that is required for the induction of myoblast differentiation.

STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release.

Cytosolic Ca(2+) signals encoded by repetitive Ca(2+) releases rely on two processes to refill Ca(2+) stores: Ca(2+) reuptake from the cytosol and activation of a Ca(2+) influx via store-operated Ca(2+) entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca(2+) sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca(2+) channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca(2+) channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca(2+) signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca(2+) sensor for their Ca(2+) homeostasis and intracellular signaling.

Human muscle economy myoblast differentiation and excitation-contraction coupling use the same molecular partners, STIM1 and STIM2.

Our recent work identified store-operated Ca(2+) entry (SOCE) as the critical Ca(2+) source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370-5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca(2+) mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca(2+) content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca(2+)-content during repeated KCl-induced Ca(2+) releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling.

STIM1- and Orai1-dependent store-operated calcium entry regulates human myoblast differentiation.

Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca(2+)](in) involving store-operated Ca(2+) entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca(2+) sensor activating Orai Ca(2+) channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca(2+)](in) and intracellular Ca(2+) store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K(+) channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.