Calcium-Inducible MAPK/AP-1 Signaling Drives Semaphorin 3A Expression in Normal Human Epidermal Keratinocytes

Abstract

Epidermal keratinocytes are primarily involved in the expression of semaphorin (Sema) 3A, which is involved in the regulation of cutaneous innervation. However, the mechanisms underlying the intracellular signaling of Sema3A expression in keratinocytes remain unknown. We herein investigated the signaling mechanisms for the induction of Sema3A expression in normal human epidermal keratinocytes (NHEKs). Sema3A expression is transiently increased in calcium-stimulated NHEKs, whereas it is markedly decreased in terminally differentiated NHEKs. Sema3A mRNA is mainly localized in the stratum basale and stratum suprabasale of the epidermis. We cloned the 5'-flanking region of the Sema3A gene and identified a critical region for Sema3A promoter activity within -134 base pairs of the start codon. We found transcription factor binding sites, including that for activator protein (AP)-1, in this region. Sema3A expression was increased by the co-overexpression of JunB and Fra-2 in the presence of 0.1 or 1.4 mM calcium. The calcium-mediated transient upregulation of Sema3A expression was significantly suppressed by mitogen-activated protein kinase (MAPK)/extracellular signaleregulated kinase (ERK) kinase (MEK) 1/2 or AP-1 inhibitors. These results demonstrate that the calciummediated transient upregulation of Sema3A in NHEKs is involved in the MEK/ERK and AP-1 signaling axis. Therefore, Sema3A mRNA may be expressed in the lower epidermis under controlled conditions by calcium via the MAPKeAP-1 axis.

Konjac Ceramide (kCer)-Mediated Signal Transduction of the Sema3A Pathway Promotes HaCaT Keratinocyte Differentiation

“…Nrp1 is upregulated in primary keratinocytes in response to differentiating media [ 13 ]. Sema3A represses neurogenesis via Nrp1, and also plays an important role in keratinocyte migration and differentiation [ 26 , 27 , 28 ]. Our previous study demonstrated that Sema3A and kCer do not affect histamine-GPCR-mediated cell migration and undifferentiation [ 12 ].…”

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

“…The culture medium was replaced with antibiotic-free medium and the cells were co-transfected with 500 ng/well pGL3-Sema3A vector containing the firefly luciferase gene and 50 ng/well pRL3-TK vector containing the Renilla luciferase gene (Promega, Madison, WI, USA) using X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer's instructions. pGL3-Sema3A vector was produced in accordance with the methods reported by Kamata et al [14]. At 24 hours after the start of transfection, the medium was replaced with fresh medium containing the test samples shown in Table 1, apigenin, baicalein, baicalin, chrysin, or wogonin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at various concentrations, and NHEKs were further incubated for 24 or 48 hours at 37˚C.…”

“…Cultured NHEKs were transfected with luciferase reporter gene constructs containing -1444 bp of the human Sema3A promoter as described previously [14]. Following transfection, cells were treated with various types of herbal plant extracts at a final concentration of 50 μg/mL for 24 hours and the cell lysates were subjected to luciferase assays.…”

“…Thus, the different structure in ring A might affect activity on the Sema3A promoter in epidermal keratinocytes, although the precise mechanisms remain unclear in this study. Recently, we reported that Sema3A mRNA expression in NHEKs is regulated by calcium via the mitogen-activated protein kinase and activator protein (AP)-1 signaling axis [14]. Meanwhile, baicalein has been shown to suppress the transcription activity of AP-1 [28].…”

Semaphorin3A Promotes the Development of Nickel Allergy

“…Sema3A is reported to mainly localize in the keratin14-positive keratinocytes in the stratum basale 127 of the human epidermis 27 . Therefore, to investigate the role of Sema3A in Ni allergy in vivo, we crossed Sema3A fl/fl mice with keratin5 (K5)-Cre mice using a cre/loxp system to knockout Sema3A in K5, which is a similar protein and partner of keratin14.…”