Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation.

Transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-ß receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

Screening quality for Ca2+-activated potassium channel in IonWorks Quattro is greatly improved by using BAPTA-AM and ionomycin.

INTRODUCTION: IonWorks automated patch clamp systems are being widely used for ion channel drug discovery, but the perforated patch mode of these systems makes it difficult to obtain a steady intracellular Ca(2+) concentration ([Ca(2+)](i)). This difficulty prevents obtaining high-quality data regarding Ca(2+)-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca(2+)](i) in the IonWorks Quattro automated patch clamp system to evaluate BK channels. METHODS: Electrophysiological recordings were performed using the single-hole or population patch clamp mode of IonWorks Quattro. To increase [Ca(2+)](i), ionomycin was used. The variation in the BK current and the effect of BK channel modulators were examined in the presence and absence of an intracellular Ca(2+) chelator, BAPTA-AM (20µM). RESULTS: BK current activated by step pulses to +100mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1Hz. Approximately 30% of cells exhibited current variation exceeding 20% when the variation was calculated using the first and third pulses. However, BAPTA-AM treatment before current measurement decreased the number of cells displaying large variation (>20%) to 5%. In the presence of BAPTA-AM, the BK channel modulators NS1619 and 12,14-dichlorodehydroabietic acid increased the BK current at concentrations of 10µM or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30µM. DISCUSSION: The main finding of this study is that the [Ca(2+)](i) variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca(2+)-activated ion channels. The application of BAPTA-AM and ionomycin greatly improved the precision of BK channel screening, and this method should be applicable to other Ca(2+)-activated ion channels such as SK channels.

Overexpression of the transcription factor Yin-Yang-1 suppresses differentiation of HaCaT cells in three-dimensional cell culture.

Yin-Yang-1 (YY1) is a member of the GLI-Krüppel family of transcription factors, and both YY1 mRNA and protein expression have been identified in a number of different tissues and cell types suggesting that it is expressed both constitutively and ubiquitously. In epidermal tissue, however, we reported previously that YY1 protein is expressed at high levels in undifferentiated basal keratinocytes and is downregulated during differentiation toward the suprabasal layers. This differential expression pattern during keratinocyte differentiation suggests that YY1 may have an important role in regulating keratinocyte differentiation. In this study, we examined the role of YY1 in differentiation of the human keratinocyte cell line HaCaT using air-liquid interface three-dimensional culture. The constitutive overexpression of YY1 in HaCaT cells during air exposure-induced differentiation resulted in an undifferentiated phenotype, thickening of the stratified layers, suppression of differentiation marker expression, and retention of proliferative activity. These findings suggested that YY1 may have an important role in maintenance of the undifferentiated phenotype of keratinocytes in the basal epidermal layer, and that reduction of YY1 expression in the suprabasal layers may allow keratinocytes to differentiate and move toward the upper layers of the epidermis.