Role of STIM1-and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation
ABSTRACT The barrier function of skin is due to the thin layer on the uppermost surface of the skin called the epidermis. The epidermis has a multilayered structure in which each layer consists of keratinocytes (KCs) of different differentiation status. The integrity of KC differentiation is critical for the function of skin and its abrogation causes or is accompanied by skin diseases. Intracellular and extracellular Ca(2+) are known to play important roles in KC differentiation. However, the molecular mechanisms underlying Ca(2+)-regulation of KC differentiation are still largely unknown. Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most non-excitable cells. SOCE is evoked in response to a fall in [Ca(2+)] in the endoplasmic reticulum (ER). Two proteins have been identified as essential components of SOCE: STIM1, a Ca(2+) sensor in the ER, and Orai1, a subunit of Ca(2+) 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 Ca(2+) concentration from low (0.03 mM; undifferentiated KCs) to high (1.8 mM; differentiated KCs). This Ca(2+) switch triggers phospholipase C-mediated intracellular Ca(2+) signals (Ca(2+)-switch-induced Ca(2+) response), which would likely involve the activation of SOCE. Knockdown of either STIM1 or Orai1 strongly suppressed SOCE and almost completely abolished the Ca(2+)-switch-induced Ca(2+) 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 Ca(2+) condition and inhibited the growth arrest in response to Ca(2+) switch. These results demonstrate that SOCE plays multiple critical roles in KC differentiation and function.
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ABSTRACT: The use of usnic acid as a weight loss agent is a safety concern due to reports of acute liver failure in humans. Previously we demonstrated that usnic acid induces apoptosis and cytotoxicity in hepatic HepG2 cells. We also demonstrated that usnic acid induces autophagy as a survival mechanism against its cytotoxicity. In this study, we investigated and characterized further molecular mechanisms underlying the toxicity of usnic acid in HepG2 cells. We found that usnic acid causes endoplasmic reticulum (ER) stress demonstrated by the increased expression of typical ER stress markers, including CHOP, ATF-4, p-eIF2α, and spliced XBP1. Usnic acid inhibited the secretion of Gaussia luciferase measured by an ER stress reporter assay. An ER stress inhibitor 4-phenylbutyrate attenuated usnic acid-induced apoptosis. Moreover, usnic acid significantly increased the cytosolic free Ca(2+) concentration. Usnic acid increased the expression of calcium release-activated calcium channel protein 1 (CRAM1 or ORAI1) and stromal interaction molecule 1 (STIM1), two key components of store-operated calcium entry (SOCE), which is the major Ca(2+) influx pathway in non-excitable cells, this finding was also confirmed in primary rat hepatocytes. Furthermore, knockdown of ORAI1 prevented ER stress and ATP depletion in response to usnic acid. In contrast, overexpression of ORAI1 increased ER stress and ATP depletion caused by usnic acid. Taken together, our results suggest that usnic acid disturbs calcium homeostasis, induces ER stress, and that usnic acid-induced cellular damage occurs at least partially via activation of the Ca(2+) channel of SOCE. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.Toxicological Sciences 04/2015; DOI:10.1093/toxsci/kfv075 · 4.48 Impact Factor
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ABSTRACT: Ca(2+) influx controls essential epidermal functions, including proliferation, differentiation, cell migration, itch, and barrier homeostasis. The Orai1 ion channel allows capacitive Ca(2+) influx after Ca(2+) release from the endoplasmic reticulum, and it has now been shown to modulate epidermal atrophy. These findings reveal new interactions among various Ca(2+) signaling pathways and uncover novel functions for Ca(2+) signaling via the Orai1 channel.Journal of Investigative Dermatology 06/2014; 134(6):1506-8. DOI:10.1038/jid.2014.54 · 6.37 Impact Factor
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ABSTRACT: Regular keratinocyte differentiation is crucial for the formation of an intact epidermal barrier and is triggered by extracellular calcium. Disturbances of epidermal barrier formation and aberrant keratinocyte differentiation are involved in the pathophysiology of several skin diseases, such as psoriasis, atopic dermatitis, basal and squamous skin cancer, and genetic skin diseases such as Darier's disease and Olmstedt syndrome. In this review, we summarize current knowledge about the underlying molecular mechanisms of calcium-induced differentiation in keratinocytes. We provide an overview of calcium's genomic and non-genomic mechanisms to induce differentiation and discuss the calcium gradient in the epidermis, giving rise to cornified skin and lipid envelope formation. We focus on the calcium-sensing receptor, transient receptor potential channels, and STIM/Orai as the major constituents of calcium sensing and calcium entry in the keratinocytes. Finally, skin diseases linked to impaired differentiation will be discussed, paying special attention to disturbed TRP channel expression and TRP channel mutations.European journal of dermatology: EJD 12/2014; DOI:10.1684/ejd.2014.2452 · 1.95 Impact Factor