Kehinde Ross

Agonist-induced calcium entry correlates with STIM1 translocation

The mechanisms of agonist‐induced calcium entry (ACE) following depletion of intracellular calcium stores have not been fully established. We report here that calcium‐independent phospholipase A (iPLA2) is required for robust Ca2+ entry in HaCaT keratinocytes following ATP or UTP stimulation. Lysophosphatidic acid (LPA), an unrelated agonist, evoked Ca2+ release without inducing robust Ca2+ entry. Both LPA and UTP induced the redistribution of STIM1 into puncta which localized to regions near or at the plasma membrane, as well as within the cytoplasm. Plasma membrane‐associated STIM1 remained high for up to 10 min after UTP stimulation, whereas it had returned almost to baseline by that time point in LPA‐stimulated cells. This correlated with faster reloading of the endoplasmic reticulum Ca2+ stores in LPA treated cells. Thus by differentially regulating store‐refilling after agonist‐mediated depletion, LPA and UTP may exert distinct effects on the duration of STIM1 localization at the plasma membrane, and thus, on the magnitude and duration of ACE. J. Cell. Physiol. 211: 569–576, 2007.

Identification of Threonine 66 as a Functionally Critical Residue of the Interleukin-1 Receptor-associated Kinase

We have mutated a conserved residue of the death domain of the interleukin-1 (IL-1) receptor-associated kinase (IRAK), threonine 66. The substitution of Thr-66 with alanine or glutamate prevented spontaneous activation of NF-κB by overexpressed IRAK but enhanced IL-1-induced activation of the factor. Like the kinase-inactivating mutation, K239S, the T66A and T66E mutations interfered with the ability of IRAK to autophosphorylate and facilitated the interactions of IRAK with TRAF6 and with the IL-1 receptor accessory protein, AcP. Wild-type IRAK constructs tagged with fluorescent proteins formed complexes that adopted a punctate distribution in the cytoplasm. The Thr-66 mutations prevented the formation of these complexes. Measurements of fluorescence resonance energy transfer among fluorescent constructs showed that the Thr-66 mutations abolished the capacity of IRAK to dimerize. In contrast, the K239S mutation did not inhibit dimerization of IRAK as evidenced by fluorescence resonance energy transfer measurements, even though microscopy showed that it prevented the formation of punctate complexes. Our results show that Thr-66 plays a crucial role in the ability of IRAK to form homodimers and that its kinase activity regulates its ability to form high molecular weight complexes. These properties in turn determine key aspects of the signaling function of IRAK.

Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca2+i Mobilization and NFAT2 Activation

Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μℳ LPA in 60 μℳ Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mℳ Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway.

Expression of microRNA-184 in keratinocytes represses argonaute 2.

Interleukin‐22 (IL‐22) is a proinflammatory cytokine that has been associated with the pathogenesis of inflammatory skin disorders. However, the impact of IL‐22 on microRNA (miRNA) expression in epidermal keratinocytes is unknown. Here we show that IL‐22 induces miR‐184 in reconstituted human epidermis (RHE) and in the HaCaT keratinocyte cell line. Exposure to IL‐22 increased miR‐184 expression 8‐ and 15‐fold in RHE and HaCaT cells, respectively. Oncostatin M, an unrelated proinflammatory cytokine, also raised miR‐184 expression in RHE and HaCaT keratinocytes. Pharmacologic and genetic inhibition demonstrated that cytokine‐induced expression of miR‐184 was mediated by signal transducer and activation of transcription 3 (STAT3). Argonaute 2 (AGO2), a member of the RNA‐induced silencing complex (RISC), is a predicted miR‐184 target. Using protein, messenger RNA and reporter analyses, we found that miR‐184 regulates the expression of AGO2. We conclude that cytokine‐induced miR‐184 attenuates AGO2 expression in keratinocytes. J. Cell. Physiol. 228: 2314–2323, 2013.

Inhibition of calcium-independent phospholipase A impairs agonist-induced calcium entry in keratinocytes.

Background  In many cells, depletion of intracellular calcium (Ca2+) reservoirs triggers Ca2+ entry through store‐operated Ca2+ channels in the plasma membrane. However, the mechanisms of agonist‐induced calcium entry (ACE) in keratinocytes are not fully understood.

Towards topical microRNA-directed therapy for epidermal disorders

Abstract There remains an unmet dermatological need for innovative topical agents that achieve better longterm outcomes with fewer side effects. Modulation of the expression and activity of microRNA (miRNAs) represents an emerging translational framework for the development of such innovative therapies because changes in the expression of one miRNA can have wide‐ranging effects on diverse cellular processes associated with disease. In this short review, the roles of miRNA in epidermal development, psoriasis, cutaneous squamous cell carcinoma and re‐epithelisation are highlighted. Consideration is given to the delivery of oligonucleotides that mimic or inhibit miRNA function using vehicles such as cell penetrating peptides, spherical nucleic acids, deformable liposomes and liquid crystalline nanodispersions. Formulation of miRNA‐directed oligonucleotides with such skin‐penetrating epidermal agents will drive the development of RNA‐based cutaneous therapeutics for deployment as primary or adjuvant therapies for epidermal disorders. Graphical abstract Figure. No Caption available.

MicroRNA-184 Is Induced By Store-Operated Calcium Entry And Regulates Early Keratinocyte Differentiation

Extracellular calcium (Ca2+) and store-operated Ca2+ entry (SOCE) govern homeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report exogenous Ca2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca2+ for 5 days. In contrast, neither phorbol ester nor 1, 25-dihydroxyvitamin D3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca2+-dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a 4-fold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a 3-fold rise in levels of cyclin E and doubled the levels of γH2AX, a marker of DNA double strand breaks. The p21 cyclin-dependent kinase (CDK) inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to a SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation.

MICRORNA-184 AND ITS LNCRNA SPONGE UCA1 ARE INDUCED IN WOUNDED KERATINOCYTES IN A STORE-OPERATED CALCIUM ENTRY-DEPENDENT MANNER

Emerging evidence implicates microRNAs (miRNA) in the regulation of keratinocyte migration. However, the putative roles of microRNA-184 (miR-184) in keratinocyte migration have not been examined. Here, we show that miR-184 expression was elevated following wounding of human keratinocyte monolayers. The induction of miR-184 was dependent on store-operated calcium entry (SOCE) as it was abolished by pharmacologic SOCE blockers. The long non-coding RNA urothelial cancer associated 1 (UCA1), which is thought to acts as a sponge or competing endogenous RNA (ceRNA) against miR-184 was also induced in scratched monolayers. Induction of UCA1 was impaired, but not abolished, by SOCE inhibition. Transfection of keratinocytes with a miR-184 mimic stimulated migration in scratch assays, whereas inhibition of miR-184 dampened the ability of keratinocytes to migrate. Together, our data suggest, for the first time, that SOCE promotes miR-184 induction in wounded monolayers to support keratinocyte migration while also increasing lncRNA UCA1 expression, which may in turn regulate miR-184 activity in keratinocytes.

Lysophosphatidic acid promotes keratinocyte migration through STIM1-regulated Orai1-Ca2+ channels and NFAT2 activation

S | Growth Factors and Signal Transduction