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Phospholipase D (PLD) has been implicated in signal transduction and membrane traffic. We have previously shown that phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) stimulates in vitro partially purified brain membrane PLD activity, defining a novel function of PtdIns-4,5-P2 as a PLD cofactor. In the present study we extend these observations to permeabilized U937 cells. In these cells, the activation of PLD by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) is greatly potentiated by MgATP. We have utilized this experimental system to test the hypothesis that MgATP potentiates PLD activation by G proteins because it is required for PtdIns-4,5-P2 synthesis by phosphoinositide kinases. As expected, MgATP was absolutely required for maintaining elevated phosphatidylinositol 4-phosphate (PtdIns-4-P) and PtdIns-4,5-P2 levels in the permeabilized cells. In the presence of MgATP, GTP gamma S further elevated the levels of the phosphoinositides. The importance of PtdIns-4,5-P2 for PLD activation was examined by utilizing a specific inhibitory antibody directed against phosphatidylinositol 4-kinase (PtdIns 4-kinase), the enzyme responsible for the first step in the synthesis of PtdIns-4,5-P2. Anti-PtdIns 4-kinase completely inhibited PtdIns 4-kinase activity in vitro and reduced by 75-80% PtdIns-4-P and PtdIns-4,5-P2 levels in the permeabilized cells. In parallel, the anti-PtdIns 4-kinase fully inhibited the activation of PLD by GTP gamma S and caused a 60% inhibition of PLD activation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, indicating that elevated PtdIns-4,5-P2 levels are required for PLD activation. This conclusion is supported by the fact that neomycin, a high affinity ligand of PtdIns-4,5-P2, also blocked PLD activation. Furthermore, the activity of PLD in U937 cell lysate was stimulated by PtdIns-4,5-P2 in a dose-dependent manner. The current results indicate that PtdIns-4,5-P2 synthesis is required for PLD activation in permeabilized U937 cells and strongly support the proposed function of PtdIns-4,5-P2 as a cofactor for PLD. In addition, the results further establish PtdIns-4,5-P2 as a key component in the generation of second messengers via multiple pathways including phosphoinositide-phospholipase C, phosphoinositide 3-kinase and PLD.

The functional role of VR1, which we and others have recently identified on several epithelial and mesenchymal human skin cell populations, was investigated in the human hair follicle (HF), as a prototypic epithelial–mesenchymal interaction system. VR1 immunoreactivity was confined to distinct epithelial compartments of HFs in anagen and catagen, while dermal papilla fibroblasts and HF melanocytes were VR1 negative. In organ culture, VR1 activation by capsaicin resulted in a dose-dependent and VR1-specific inhibition of hair shaft elongation, suppression of proliferation, promotion of apoptosis, and induction of catagen transformation, possibly due to upregulation of a potent hair growth inhibitor TGFβ2. Cultured outer root sheath (ORS), as well as HaCaT, keratinocytes also expressed functional VR1, whose stimulation inhibited proliferation, induced apoptosis, and elevated intracellular calcium concentration. Finally, VR1 stimulation of cultured ORS keratinocytes upregulated the expression of recognized endogenous hair growth inhibitors (IL-1β and TGFβ2) and downregulated the expression of stimulators (HGF, IGF-1, and SCF), while key differentiation markers (CK17, CK14, filaggrin, and involucrin) remained unaffected. In conclusion, VR1 is a significant novel player in human hair growth control underscoring that its physiological functions in human skin far extend beyond sensory neuron-coupled nociception.

Neurotrophins are important modulators of epithelial-mesenchymal interactions. Previously, we had shown that brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tyrosine kinase B (TrkB) are prominently involved in the control of murine hair follicle cycling. We now show that BDNF and TrkB are also expressed in the human hair follicle in a manner that is both hair cycle dependent and suggestive of epithelial-mesenchymal cross-talk between BDNF-secreting dermal papilla fibroblasts of anagen hair follicles and subpopulations of TrkB+ hair follicle keratinocytes. As functional evidence for an involvement of BDNF/TrkB in human hair growth control, we show in organ-cultured human anagen hair follicles that 50 ng per mL BDNF significantly inhibit hair shaft elongation, induce premature catagen development, and inhibit keratinocyte proliferation. Quantitative real-time rtPCR analysis demonstrates upregulation of the potent catagen inducer, transforming growth factor beta2 (TGFbeta2) by BDNF, whereas catagen induction by BDNF was partially reversible through co-administration of TGFbeta-neutralizing antibody. This suggests that TrkB-mediated signaling promotes the switch between anagen and catagen at least in part via upregulation of TGFbeta2. Thus, human scalp hair follicles are both a source and target of bioregulation by BDNF, which invites to target TrkB-mediated signaling for therapeutic hair growth modulation.

Background After implementation of the PREDICA annual chest X-ray (CXR) screening program in smokers in the general practice setting of Varese-Italy a significant reduction in lung cancer-specific mortality (18 %) was observed. The objective of this study covering July 1997 through December 2006 was to estimate the cost-effectiveness of this intervention. Methods We examined detailed information on lung cancer (LC) cases that occurred among smokers invited to be screened in the PREDICA study (Invitation-to-screening Group, n = 5815 subjects) to estimate costs and quality-adjusted life-years (QALYs) from LC diagnosis until death. The control group consisted of 156 screening-eligible smokers from the same area, uninvited and unscreened, who developed LC and were treated by usual care. We calculated the incremental net monetary benefit (INMB) by comparing LC management in screening participants (n = 1244 subjects) and in the Invitation-to-screening group versus control group. Results The average number of QALYs since LC diagnosis was 1.7, 1.49 and 1.07, respectively, in screening participants, the invitation-to-screening group, and the control group. The average total cost (screening + management) per LC case was higher in screening participants (€17,516) and the Invitation-to-screening Group (€16,167) than in the control group (€15,503). Assuming a maximum willingness to pay of €30,000/QALY, we found that the intervention was cost-effective with high probability: 79 % for screening participation (screening participants vs. control group) and 95 % for invitation-to-screening (invitation-to-screening group vs. control group). Conclusions Based on the PREDICA study, annual CXR screening of high-risk smokers in a general practice setting has high probability of being cost-effective with a maximum willingness to pay of €30,000/QALY.