Modulation of semaphorin 3A expression by calcium concentration and histamine in human keratinocytes and fibroblasts
Introduction
C-fiber is known as a primary afferent nerve to deliver itch to the central nerve system [1]. Numerical increment and elevated sprouting of C-fiber in the epidermis easily produce itch and accelerate the itch–scratch cycle [2]. In addition, C-fiber plays pluripotential roles for itch-related events. For example, neuropeptides released from C-fibers stimulate keratinocytes and mast cells to produce pruritogenic factors that again stimulate C-fiber [3], [4], [5].
Both neurotrophins and chemorepellents regulate the elongation and sprouting of itch-associated C-fibers in the skin. Nerve growth factor (NGF) [6], [7] and semaphorin 3A (Sema3A) [8] are representatives of these two types of nerve axon-guidance factors, respectively. NGF and Sema3A have opposite effects on C-fiber elongation, which is related to promotion of itch in the skin. In atopic dermatitis, the number of peripheral nerve endings is increased and may amplify itch [2]. NGF is known to initiate the sprouting of epidermal nerve fibers in atopic dermatitis and its model NC/Nga mice [9]. Changes in Sema3A expression/production has also been reported in the lesional skin of atopic dermatitis [10], [11]. A reduction in Sema3A expression allows nerve fibers to sprout into the upper epidermis, and photochemotherapy may exert a therapeutic effect by increasing epidermal cell production of Sema3A [12]. On the other hands, in normal skin, the extracellular calcium concentration critically determines a variety of biological activities of keratinocytes [13]. The calcium concentration increases towards the outer epidermis, forming a “calcium gradient” within the epidermis [14]. It is possible that this physiological condition influences on the production of the nerve guidance factors. In addition, histamine released from mast cells in certain allergic conditions possibly modulates their production.
To address the regulatory mechanisms underlying C-fiber elongation, we investigated the effects of calcium concentration on the expression of NGF and SEMA3A in normal human epidermal keratinocytes (NHEK). Upon allergic stimulation, histamine and tryptase are produced by mast cells, and keratinocytes express H1 receptors for histamine and protease-activated receptor-2 (PAR-2) for tryptase [15], [16]. Therefore, it is an interesting issue to investigate the effects of histamine and a PAR-2 agonist, SLIGRL-NH2 (SLIGRL) on the expression of these guidance factors in NHEK and normal human fibroblasts (NHFb). Results suggest that the physiological and pathological conditions of the skin modulate the production of NGF and Sema3A by keratinocytes and fibroblasts.
Section snippets
Chemicals
Histamine and a PAR-2 agonist SLIGRL were purchased from Wako (Osaka, Japan) and Tocris (Ellisville, MO), respectively. These substances were freshly diluted by distilled water before use.
Cell and cell culture
Primary culture of NHEK was obtained from Lonza (Basel, Switzerland). NHEK were grown in Keratinocyte Growth Medium-2 (KGM-2) for expansion and Keratinocyte Basal Medium-2 (KBM-2; Lonza) for experiments at 37 °C in a 5% CO2 atmosphere. Primary NHFb were obtained from Takara (Osaka, Japan) and cultured in
High calcium augments the expression of SEMA3A but not NGF in NHEK
Keratinocytes proliferate at a low calcium concentration, such as 0.15 mM, and differentiate at a high calcium concentration in culture [12]. The proliferating and differentiating cells represent the basal cell in the lower most epidermis and the prickle cell in the upper epidermis, respectively. To address the physiological production of Sema3A and NGF by epidermal keratinocytes, NHEK were cultured under varying calcium concentrations, ranging from 0.15 to 0.9 mM, and after 2-h incubation, the
Discussion
In this study, we demonstrated the modulation of SEMA3A and NGF expressions by calcium concentration and histamine or SLIGRL. The expression of SEMA3A depended on calcium concentration in NHEK, but not in NHFb. Whereas NHEK cultured at low calcium concentrations of 0.15–0.3 mM, inducible for keratinocyte proliferation, expressed low levels of SEMA3A, cells cultured at high calcium concentrations of 0.45–0.75 mM, suitable for keratinocyte differentiation, expressed high levels of SEMA3A. On the
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