SummaryIntracellular Ca2+ signals constitute key elements in signal transduction. Of the three major Ca2+ mobilizing messengers described, the most potent, nicotinic acid adenine dinucleotide phosphate (NAADP) is the least well understood in terms of its molecular targets [1]. Recently, we showed that heterologous expression of two-pore channel (TPC) proteins enhances NAADP-induced Ca2+ release, whereas the NAADP response was abolished in pancreatic beta cells from Tpcn2 gene knockout mice [2]. However, whether TPCs constitute native NAADP receptors is unclear. Here we show that immunopurified endogenous TPC complexes possess the hallmark properties ascribed to NAADP receptors, including nanomolar ligand affinity [3–5]. Our study also reveals important functional differences between the three TPC isoforms. Thus, TPC1 and TPC2 both mediate NAADP-induced Ca2+ release, but the subsequent amplification of this trigger Ca2+ by IP3Rs is more tightly coupled for TPC2. In contrast, TPC3 expression suppressed NAADP-induced Ca2+ release. Finally, increased TPC expression has dramatic and contrasting effects on endolysosomal structures and dynamics, implicating a role for NAADP in the regulation of vesicular trafficking. We propose that NAADP regulates endolysosomal Ca2+ storage and release via TPCs and coordinates endoplasmic reticulum Ca2+ release in a role that impacts on Ca2+ signaling in health and disease [6].

In whole-cell recordings from HaCaT keratinocytes, ATP, bradykinin, and histamine caused a biphasic change of the membrane potential consisting of an initial transient depolarization, followed by a pronounced and long-lasting hyperpolarization. Flash photolysis of caged IP3 mimicked the agonist-induced voltage response, suggesting that intracellular Ca2+ release and subsequent opening of Ca2+-activated ion channels serve as the common transduction mechanism. In contrast, cAMP- and PKC-dependent pathways were not involved in the electrophysiological effects of the extracellular signaling molecules. The depolarization was predominantly mediated by a DIDS- and niflumic acid-sensitive Cl- current, whereas a charybdotoxin- and clotrimazole-sensitive K+ current underlay the prominent hyperpolarization. Consistent with the electrophysiological data, RT-PCR showed that HaCaT keratinocytes express two types of Ca2+-activated Cl- channels, CaCC2 and CaCC3 (CLCA2), as well as the Ca2+-activated K+ channel hSK4. That the pronounced hSK4-mediated hyperpolarization bears significance on the growth and differentiation properties of keratinocytes is suggested by RNase protection assays showing that hSK4 mRNA expression is strongly down-regulated under conditions that allow keratinocyte differentiation. hSK4 might thus play a role in linking changes in membrane potential to the biological fate of keratinocytes.

Insulin-like growth factor 1 (IGF-1) is an important regulator of growth, survival, and differentiation in many tissues. It is produced in several isoforms that differ in their N-terminal signal peptide and C-terminal extension peptide. The locally acting isoform of IGF-1 (mIGF-1) was previously shown to enhance the regeneration of both muscle and heart. In this study, we tested the therapeutic potential of mIGF-1 in the skin by generating a transgenic mouse model in which mIGF-1 expression is driven by the keratin 14 promoter. IGF-1 levels were unchanged in the sera of hemizygous K14/mIGF-1 transgenic animals whose growth was unaffected. A skin analysis of young animals revealed normal architecture and thickness as well as proper expression of differentiation and proliferation markers. No malignant tumors were formed. Normal homeostasis of the putative stem cell compartment was also maintained. Healing of full-thickness excisional wounds was accelerated because of increased proliferation and migration of keratinocytes, whereas inflammation, granulation tissue formation, and scarring were not obviously affected. In addition, mIGF-1 promoted late hair follicle morphogenesis and cycling. To our knowledge, this is the first work to characterize the simultaneous, stimulatory effect of IGF-1 delivery to keratinocytes on two types of regeneration processes within a single mouse model. Our analysis supports the use of mIGF-1 for skin and hair regeneration and describes a potential cell type-restricted action. (Am J

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis. IntroductionThe outermost layer of the skin is formed by the epidermis, a stratified squamous epithelium mainly consisting of keratinocytes. A remarkable property of the epidermis is its self-renewing capacity. Keratinocytes of the basal layer proliferate to generate new cells that differentiate and move upward. Terminally differentiated and metabolically inactive cells form a tightly linked sheet; they are continuously sloughed off from the surface and replaced by cells from the underlying layers (1). To maintain the balance between proliferation and differentiation, a tight spatial and temporal regulation of gene expression is essential. Therefore, it is of particular interest to identify and functionally characterize the transcription factors involved in the control of epidermal homeostasis.A promising new candidate in this regulation is serum response factor (SRF), a member of the highly conserved MADS box family of transcription factors (2, 3). SRF regulates the expression of immediate early genes (3-5) and of muscle-specific genes (reviewed in ref. 6) after binding to its target sequence (CArG box), which is often located within a serum response element. In fibroblasts, SRF-dependent gene transcription is controlled by 2 types of cofactors that are regulated by different signaling pathways. Cofactors of the myocardin-related transcription factor (MRTF) family can respond to Rho family GTPases and monomeric actin. The second class of SRF

Regulated cell proliferation is a crucial prerequisite for Schwann cells to achieve myelination in development and regeneration. In the present study, we have investigated the function of the cell cycle inhibitors p21 and p16 as potential regulators of Schwann cell proliferation, using p21- or p16-deficient mice. We report that both inhibitors are required for proper withdrawal of Schwann cells from the cell cycle during development and following injury. Postnatal Schwann cells express p21 exclusively in the cytoplasm, first detectable at postnatal day 7. This cytoplasmic p21 expression is necessary for proper Schwann cell proliferation control in the late development of peripheral nerves. After axonal damage, p21 is found in Schwann cell nuclei during the initiation of the proliferation period. This stage is critically regulated by p21, since loss of p21 leads to a strong increase in Schwann cell proliferation. Unexpectedly, p21 levels are upregulated in this phase suggesting that the role of p21 may be more complex than purely inhibitory for the Schwann cell cycle. However, inhibition of Schwann cell proliferation is the overriding crucial function of p21 and p16 in peripheral nerves as revealed by the consequences of loss-of-function in development and after injury. Different mechanisms appear to underlie the inhibitory function, depending on whether p21 is cytoplasmic or nuclear.