The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3 has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

Pulmonary vascular medial hypertrophy caused by excessive pulmonary artery smooth muscle cell (PASMC) proliferation is a major cause for the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased Ca 2؉ influx is an important stimulus for PASMC proliferation. Transient receptor potential (TRP) channel genes encode Ca 2؉ channels that are responsible for Ca 2؉ entry during cell proliferation. Normal human PASMC expressed multiple canonical TRP (TRPC) isoforms; TRPC6 was highly expressed and TRPC3 was minimally expressed. The protein expression of TRPC6 in normal PASMC closely correlated with the expression of Ki67, suggesting that TRPC6 expression is involved in the transition of PASMC from quiescent phase to mitosis. In lung tissues and PASMC from IPAH patients, the mRNA and protein expression of TRPC3 and -6 were much higher than in those from normotensive or secondary pulmonary hypertension patients. Inhibition of TRPC6 expression with TRPC6 small interfering RNA markedly attenuated IPAH-PASMC proliferation. These results demonstrate that expression of TRPC channels correlates with the progression of the cell cycle in PASMC. TRPC channel overexpression may be partially responsible for the increased PASMC proliferation and pulmonary vascular medial hypertrophy in IPAH patients.I diopathic pulmonary arterial hypertension (IPAH) is a fatal disease that causes right heart failure and death. The elevated pulmonary vascular resistance (PVR) and arterial pressure in IPAH patients result mainly from pulmonary vasoconstriction, vascular remodeling, and in situ thrombosis (1). A central aspect of pulmonary vascular remodeling is medial hypertrophy caused by sustained pulmonary vasoconstriction (2-4), excessive pulmonary artery smooth muscle cell (PASMC) proliferation (5), and inhibited PASMC apoptosis (6, 7), resulting in a narrowed vascular lumen and increased PVR. Although its etiology remains unclear, elevated levels of circulating mitogens, dysfunction or down-regulation of receptors and ion channels, upregulation of transporters, and heightened activity of elastases and glycoproteins have been implicated in IPAH (5,6,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) Transient receptor potential (TRP) channel genes may encode subunits that form receptor-(ROC) and store-(SOC) operated Ca 2ϩ channels in many cell types, including PASMC and pulmonary artery endothelial cells (PAEC) (28,(30)(31)(32)(33)(34). Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21). High levels of [Ca 2ϩ ] cyt and sufficient levels of Ca 2ϩ in the SR are required for vascular smooth muscle cell proliferation (22,25,39). Because they regulate SR and cytoplasmic Ca 2ϩ , CCE and SOC may play significant roles in regulating cell proliferation (28,29). This study tested the hypothesis that canonical TRP (TRPC...

Pulmonary vascular medial hypertrophy in primary pulmonary hypertension (PPH) is mainly caused by increased proliferation and decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Mutations of the bone morphogenetic protein (BMP) receptor type II (BMP-RII) gene have been implicated in patients with familial and sporadic PPH. The objective of this study was to elucidate the apoptotic effects of BMPs on normal human PASMCs and to examine whether BMP-induced effects are altered in PASMCs from PPH patients. Using RT-PCR, we detected six isoforms of BMPs (BMP-1 through -6) and three subunits of BMP receptors (BMP-RIa, -RIb, and -RII) in PASMCs. Treatment of normal PASMCs with BMP-2 or -7 (100-200 nM, 24-48 h) markedly increased the percentage of cells undergoing apoptosis. The BMP-2-mediated apoptosis in normal PASMCs was associated with a transient activation or phosphorylation of Smad1 and a marked downregulation of the antiapoptotic protein Bcl-2. In PASMCs from PPH patients, the BMP-2- or BMP-7-induced apoptosis was significantly inhibited compared with PASMCs from patients with secondary pulmonary hypertension. These results suggest that the antiproliferative effect of BMPs is partially due to induction of PASMC apoptosis, which serves as a critical mechanism to maintain normal cell number in the pulmonary vasculature. Inhibition of BMP-induced PASMC apoptosis in PPH patients may play an important role in the development of pulmonary vascular medial hypertrophy in these patients.

[Ca 2ϩ ]cyt in pulmonary artery endothelial cells (PAEC). Using combined molecular biological, fluorescence microscopy, and biophysical approaches, we examined the effect of chronic hypoxia (3% O 2, 72 h) on AP-1 DNA binding activity, CCE, and transient receptor potential (TRP) gene expression in human (h) PAEC. EMSA showed that AP-1 binding to hPAEC nuclear protein extracts was significantly enhanced by hypoxia, the increase being dependent on storeoperated Ca 2ϩ influx and sensitive to La 3ϩ , an SOC inhibitor. Hypoxia also increased basal [Ca 2ϩ ]cyt, the amount of CCE produced by store depletion with cyclopiazonic acid, and the amplitude of SOC-mediated currents (I SOC). The increases of CCE amplitude and I SOC current density by hypoxia were paralleled by enhanced TRPC4 mRNA and protein expression. Hypoxia-enhanced CCE and TRPC4 expression were also attenuated by La 3ϩ . These data suggest that hypoxia increases AP-1 binding activity by enhancing Ca 2ϩ influx via La 3ϩ -sensitive TRP-encoded SOC channels in hPAEC. The Ca 2ϩ -mediated increase in AP-1 binding may play an important role in upregulating AP-1-responsive gene expression, in stimulating pulmonary vascular cell proliferation and, ultimately, in pulmonary vascular remodeling in patients with hypoxia-mediated pulmonary hypertension. activating protein-1; store-operated channels; transient receptor potential genes PULMONARY VASCULAR REMODELING in patients and animals with hypoxia-mediated pulmonary hypertension is characterized by pulmonary arterial medial hyper-