The significance of CYP11A1 expression in skin physiology and pathology.

CYP11A1, a member of the cytochrome P450 family, plays several key roles in the human body. It catalyzes the first and rate-limiting step in steroidogenesis, converting cholesterol to pregnenolone. Aside from the classical steroidogenic tissues such as the adrenals, gonads and placenta, CYP11A1 has also been found in the brain, gastrointestinal tract, immune systems, and finally the skin. CYP11A1 activity in the skin is regulated predominately by StAR protein and hence cholesterol levels in the mitochondria. However, UVB, UVC, CRH, ACTH, cAMP, and cytokines IL-1, IL-6 and TNFα can also regulate its expression and activity. Indeed, CYP11A1 plays several critical roles in the skin through its initiation of local steroidogenesis and specific metabolism of vitamin D, lumisterol, and 7-dehydrocholesterol. Products of these pathways regulate the protective barrier and skin immune functions in a context-dependent fashion through interactions with a number of receptors. Disturbances in CYP11A1 activity can lead to skin pathology.

New effects of caffeine on corticotropin-releasing hormone (CRH)-induced stress along the intrafollicular classical hypothalamic-pituitary-adrenal (HPA) axis (CRH-R1/2, IP3 -R, ACTH, MC-R2) and the neurogenic non-HPA axis (substance P, p75NTR and TrkA) in ex vivo human male androgenetic scalp hair follicles.

BACKGROUND: Human hair is highly responsive to stress, and human scalp hair follicles (HFs) contain a peripheral neuroendocrine equivalent of the systemic hypothalamic-pituitary-adrenal (HPA) stress axis. Androgenetic alopecia (AGA) is supposed to be aggravated by stress. We used corticotropin-releasing hormone (CRH), which triggers the HPA axis, to induce a stress response in human ex vivo male AGA HFs. Caffeine is known to reverse testosterone-mediated hair growth inhibition in the same hair organ culture model. OBJECTIVES: To investigate whether caffeine would antagonize CRH-mediated stress in these HFs. METHODS: HFs from balding vertex area scalp biopsies of men affected by AGA were incubated with CRH (10-7 mol L-1 ) with or without caffeine (0·001% or 0·005%). RESULTS: Compared to controls, CRH significantly enhanced the expression of catagen-inducing transforming growth factor-ß2 (TGF-ß2) (P < 0·001), CRH receptors 1 and 2 (CRH-R1/2) (P < 0·01), adrenocorticotropic hormone (ACTH) (P < 0·001) and melanocortin receptor 2 (MC-R2) (P < 0·001), and additional stress-associated parameters, substance P and p75 neurotrophin receptor (p75NTR ). CRH inhibited matrix keratinocyte proliferation and expression of anagen-promoting insulin-like growth factor-1 (IGF-1) and the pro-proliferative nerve growth factor receptor NGF-tyrosine kinase receptor A (TrkA). Caffeine significantly counteracted all described stress effects and additionally enhanced inositol trisphosphate receptor (IP3 -R), for the first time detected in human HFs. CONCLUSIONS: These findings provide the first evidence in ex vivo human AGA HFs that the stress mediator CRH induces not only a complex intrafollicular HPA response, but also a non-HPA-related stress response. Moreover, we show that these effects can be effectively antagonized by caffeine. Thus, these data strongly support the hypothesis that stress can impair human hair physiology and induce hair loss, and that caffeine may effectively counteract stress-induced hair damage and possibly prevent stress-induced hair loss.

Pregnancy-driven cardiovascular maternal miR-29 plasticity in obesity.

BACKGROUND: Obesity in pregnancy (MO) is a risk factor for maternal and/or fetal cardiovascular system disorders. This study evaluated maternal CVS expression of microRNA-29 family and its target molecules in MO to test the hypotheses: CVS miR-29 concentrations are increased in pregnancy and decreased in MO. METHODS: Non-pregnant (n=4), pregnant obese (POb, n=4), and pregnant non-obese (PnOb, n=4) baboons (Papio spp.) were studied. Maternal left ventricle (LV), left atrium (LA), and aortic arch (AA) were collected at the end of gestation. Expression of MiR-29 and elastin (ELN) mRNA were quantified. RESULTS: LA miR-29 (a, c) expression was highest in PnOb. In the LV, miR-29b expression trended lower (P=.059) for PnOb animals. ELN mRNA expression correlated positively with miR-29b expression in AA (r=.76, P=.03). CONCLUSION: Maternal obesity diminishes miR-29 adaptation to pregnancy. Pharmacologic, tissue-specific targeting of miRNA-29 may represent a strategy for prevention and treatment of MO complications.

Differential antitumor effects of vitamin D analogues on colorectal carcinoma in culture.

Colorectal cancer (CRC) is an emerging global problem with the rapid increase in its incidence being associated with an unhealthy lifestyle. Epidemiological studies have shown that decreased levels of vitamin D3 significantly increases the risk of CRC. Furthermore, negative effects of vitamin D3 deficiency can be compensated by appropriate supplementation. Vitamin D3 was shown to inhibit growth and induce differentiation of cancer cells, however, excessive vitamin D3 intake leads to hypercalcemia. Thus, development of efficient vitamin D3 analogues with limited impact on calcium homeostasis is an important scientific and clinically relevant task. The aims of the present study were to compare the antiproliferative potential of classic vitamin D3 metabolites (1α,25(OH)2D3 and 25(OH)D3) with selected low calcemic analogues (calcipotriol and 20(OH)D3) on CRC cell lines and to investigate the expression of vitamin D-related genes in CRC cell lines and clinical samples. Vitamin D3 analogues exerted anti-proliferative effects on all CRC cell lines tested. Calcipotriol proved to be as potent as 1α,25(OH)2D3 and had more efficacy than 20-hydroxyvitamin D3. In addition, the analogs tested effectively inhibited the formation of colonies in Matrigel. The expression of genes involved in 1α,25(OH)2D3 signaling and metabolism varied in cell lines analysed, which explains in part their different sensitivities to the various analogues. In CRC biopsies, there was decreased VDR expression in tumor samples in comparison to the surgical margin and healthy colon samples (p<0.01). The present study indicates that vitamin D3 analogues which have low calcemic activity, such as calcipotriol or 20(OH)D3, are very promising candidates for CRC therapy. Moreover, expression profiling of vitamin D-related genes is likely to be a powerful tool in the planning of anticancer therapy. Decreased levels of VDR and increased CYP24A1 expression in clinical samples underline the importance of deregulation of vitamin D pathways in the development of CRC.

The role of melanogenesis in regulation of melanoma behavior: melanogenesis leads to stimulation of HIF-1α expression and HIF-dependent attendant pathways.

To study the effect of melanogenesis on HIF-1α expression and attendant pathways, we used stable human and hamster melanoma cell lines in which the amelanotic vs. melanotic phenotypes are dependent upon the concentration of melanogenesis precursors in the culture media. The induction of melanin pigmentation led to significant up-regulation of HIF-1α, but not HIF-2α, protein in melanized cells for both lines. Similar upregulation of nuclear HIF-1α was observed in excisions of advanced melanotic vs. amelanotic melanomas. In cultured cells, melanogenesis also significantly stimulated expression of classical HIF-1-dependent target genes involved in angiogenesis and cellular metabolism, including glucose metabolism and stimulation of activity of key enzymes in the glycolytic pathway. Several other stress related genes containing putative HRE consensus sites were also upregulated by melanogenesis, concurrently with modulation of expression of HIF-1-independent genes encoding for steroidogenic enzymes, cytokines and growth factors. Immunohistochemical studies using a large panel of pigmented lesions revealed that higher levels of HIF-1α and GLUT-1 were detected in advanced melanomas in comparison to melanocytic nevi or thin melanomas localized to the skin. However, the effects on overall or disease free survival in melanoma patients were modest or absent for GLUT-1 or for HIF-1α, respectively. In conclusion, induction of the melanogenic pathway leads to robust upregulation of HIF-1-dependent and independent pathways in cultured melanoma cells, suggesting a key role for melanogenesis in regulation of cellular metabolism.

Endocannabinoid crosstalk between placenta and maternal fat in a baboon model (Papio spp.) of obesity.

INTRODUCTION: Maternal obesity (MO) remains a serious obstetric problem with acute and chronic morbidities for both mothers and offspring. The mechanisms underlying these adverse consequences of MO remain unknown. Endocannabinoids (ECB) are neuromodulatory lipids released from adipocytes and other tissues. Metabolic crosstalk between placenta and adipocytes may mediate sequelae of MO. The goal of this study was to elucidate placental and systemic ECB in MO. MATERIAL AND METHODS: Placentas, sera, and subcutaneous fat were collected at Cesarean sections performed near term (0.9 G) in four non-obese (nOB) and four obese (OB) baboons (Papio spp.). Concentrations of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by liquid chromatography coupled to tandem mass spectrometry. AEA and 2-AG pathways were characterized in placentas by Q-RT-PCR, Western blot and immunohistochemistry. RESULTS: Placental 2-AG levels were lower and maternal fat AEA levels were higher in OB (1254.1 ± 401.3 nmol/kg and 17.3 ± 4 nmol/kg) vs. nOB (3124.2 ± 557.3 nmol/kg and 3.1 ± 0.6 nmol/kg) animals. Concentrations of 2-AG correlated positively between maternal fat and placenta (r = 0.82, p = 0.013), but correlated negatively with maternal leptin concentrations (r = -0.72, p = 0.04 and r = -0.83, p = 0.01, respectively). CONCLUSION: This is the first study to demonstrate differential ECB pathway regulation in maternal fat and placenta in MO. Differential regulation and function exist for AEA and 2-AG as the major ECB pathways in placenta.

Ultraviolet radiation regulates cortisol activity in a waveband-dependent manner in human skin ex vivo.

BACKGROUND: 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1), 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), and glucocorticoids (GC) and their receptor (GR) play a key role in tissue-specific regulation of GC action. OBJECTIVES: To determine the expression of genes encoding 11ß-HSD1 (HSD11B1), 11ß-HSD2 (HSD11B2) and GR (GRα; also known as NC3R1) and their protein products, and levels of cortisol in human skin explants and/or cocultured keratinocytes/melanocytes after treatment with ultraviolet (UV) A, B or C wavebands. METHODS: Skin from foreskins and/or cocultured human keratinocytes/melanocytes were irradiated with UVA, UVB or UVC (skin) and incubated for 12 and 24 h. Methods of reverse transcription-polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay and immunohistochemistry (IHC) were used to determine expression and localization of corresponding genes or antigens. RESULTS: UVB enhanced the HSD11B1 gene and protein expression in a dose-dependent manner, while UVA had no effect. Similarly, UVC increased 11ß-HSD1 protein product as measured by IHC. UVB and UVC enhanced cortisol production and decreased epidermal GR expression, while UVA had no detectable effects. Although both UVA and UVB stimulated HSD11B2 gene expression, only UVA increased 11ß-HSD2 protein product levels with UVB and UVC having no effect. CONCLUSIONS: We suggest that these differential, waveband-dependent effects of UV radiation on the expression of cutaneous HSD11B1, HSD11B2 and GRα genes and their corresponding protein products, and cortisol production are to protect and/or restore the epidermal barrier homeostasis against disruption caused by the elevated cortisol level induced by UVB and UVC.

High basal NF-κB activity in nonpigmented melanoma cells is associated with an enhanced sensitivity to vitamin D3 derivatives.

BACKGROUND: Melanoma is highly resistant to current modalities of therapy, with the extent of pigmentation playing an important role in therapeutic resistance. Nuclear factor-κB (NF-κB) is constitutively activated in melanoma and can serve as a molecular target for cancer therapy and steroid/secosteroid action. METHODS: Cultured melanoma cells were used for mechanistic studies on NF-κB activity, utilising immunofluorescence, western blotting, EMSA, ELISA, gene reporter, and estimated DNA synthesis assays. Formalin-fixed, paraffin-embedded specimens from melanoma patients were used for immunocytochemical analysis of NF-κB activity in situ. RESULTS: Novel 20-hydroxyvitamin (20(OH)D(3)) and classical 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) secosteroids inhibited melanoma cell proliferation. Active forms of vitamin D were found to inhibit NF-κB activity in nonpigmented cells, while having no effect on pigmented cells. Treatment of nonpigmented cells with vitamin D3 derivatives inhibited NF-κB DNA binding and NF-κB-dependent reporter assays, as well as inhibited the nuclear translocation of the p65 NF-κB subunit and its accumulation in the cytoplasm. Moreover, analysis of biopsies of melanoma patients showed that nonpigmented and slightly pigmented melanomas displayed higher nuclear NF-κB p65 expression than highly pigmented melanomas. CONCLUSION: Classical 1,25(OH)(2)D(3) and novel 20(OH)D(3) hydroxyderivatives of vitamin D3 can target NF-κB and regulate melanoma progression in nonpigmented melanoma cells. Melanin pigmentation is associated with the resistance of melanomas to 20(OH)D(3) and 1,25(OH)(2)D(3) treatment.

Stress and the skin.

Emotional stress can affect, reveal or even exacerbate a number of skin disorders including psoriasis, atopic dermatitis, pruritus, alopecia areata, lichen planus, seborrheic dermatitis, rosacea or urticaria, although the direct pathophysiologic link between stress factors and cutaneous disease manifestation remains unclear. However, there is an increasing evidence that stress influences disease processes and contributes to the inflammation through modulating hypothalamic-pituitary-adrenal axis and releasing neuropeptides, neurotrophins, lymphokines and other chemical mediators from nerve endings and dermal cells. The central role in cellular skin reactivity to various stressors might be attributed to dermal mast cells, as they show close connections with sensory nerve endings and may release a huge number of proinflammatory mediators. However, many other cells also actively take part in skin response to stress. Although our knowledge is still not complete, one of the most distinct aspect is that the skin, endocrine, nervous and immune systems cannot longer be treated autonomously, but have to be considered as a large multidirectional complex of which interacting nature is still poorly understood.

What are melanocytes really doing all day long...?

Everyone knows and seems to agree that melanocytes are there to generate melanin - an intriguing, but underestimated multipurpose molecule that is capable of doing far more than providing pigment and UV protection to skin (1). What about the cell that generates melanin, then? Is this dendritic, neural crest-derived cell still serving useful (or even important) functions when no-one looks at the pigmentation of our skin and its appendages and when there is essentially no UV exposure? In other words, what do epidermal and hair follicle melanocytes do in their spare time - at night, under your bedcover? How much of the full portfolio of physiological melanocyte functions in mammalian skin has really been elucidated already? Does the presence or absence of melanocytes matter for normal epidermal and/or hair follicle functions (beyond pigmentation and UV protection), and for skin immune responses? Do melanocytes even deserve as much credit for UV protection as conventional wisdom attributes to them? In which interactions do these promiscuous cells engage with their immediate epithelial environment and who is controlling whom? What lessons might be distilled from looking at lower vertebrate melanophores and at extracutaneous melanocytes in the endeavour to reveal the 'secret identity' of melanocytes? The current Controversies feature explores these far too infrequently posed, biologically and clinically important questions. Complementing a companion viewpoint essay on malignant melanocytes (2), this critical re-examination of melanocyte biology provides a cornucopia of old, but under-appreciated concepts and novel ideas on the slowly emerging complexity of physiological melanocyte functions, and delineates important, thought-provoking questions that remain to be definitively answered by future research.

Modulation of corticotropin releasing factor (CRF) signaling through receptor splicing in mouse pituitary cell line AtT-20--emerging role of soluble isoforms.

Previously, using cultured human epidermal keratinocytes we have demonstrated that the activity of CRF1 receptor can be modulated by the process of alternative splicing. This phenomenon has been further investigated in the mouse corticotroph AtT-20 cell line. In the cells, transiently transfected with the plasmids coding human CRF1 isoforms, only isoforms alpha and c have shown expression on the cell membrane. Other isoforms d, e, g and h had intracellular localization with the isoform e also found in the nucleus. Co-expression of the CRF1alpha (main form of the receptor) with isoforms d, f and g prevented its expression on the cell surface resulting in accumulation of CRF1alpha inside of the cell. s expected, CRF stimulated time and dose dependent activation of CRE, CARE, AP-1 transcription elements and POMC promoter in AtT-20 cells overexpressing human CRF1alpha, while having no effect on the AP-1 transcriptional activity in cells transfected with other isoforms (d, f, g and h). However, when cells were co-transfected with CRF1alpha and CRF1e or h the CRF stimulated transcriptional activity of CRE and AP-1 was amplified in comparison to the cells expressing solely CRF1alpha; the effect was more pronounced for CRF1h than for CRF1e. In contrast, the conditioned media from the cells overexpressing CRF1e and h inhibited the CRF induced transcriptional activity in cells overexpressing CRF1alpha. Media from cells expressing CRF1h were significantly more potent that from cells transfected with CRF1e. In summary, we have demonstrated that alternatively spliced CRF1 isoforms can regulate the cellular localization of CRF1alpha, and that soluble CRF1 isoforms can have a dual effect on CRF1alpha activity depending on the intracellular vs. extracellular localization.

Vitiligo pathogenesis: autoimmune disease, genetic defect, excessive reactive oxygen species, calcium imbalance, or what else?

The pathobiology of vitiligo has been hotly disputed for as long as one remembers, and has been a magnet for endless speculation. Evidently, the different schools of thought--ranging, e.g. from the concept that vitiligo essentially is a free-radical disorder to that of vitiligo being a primary autoimmune disease--imply very different consequences for the best therapeutic strategies that one should adopt. As a more effective therapy for this common, often disfiguring pigmentary disorder is direly needed, we must strive harder to settle the pathogenesis debate definitively--on the basis of sound experimental evidence, rather than by a war of dogmatic theories. Recognizing, however, that it is theories which tend to guide our experimental designs and choice of study parameters, the various pathogenesis theories on the market deserve to be critically, yet unemotionally re-evaluated. This Controversies feature invites you to do so, and to ask yourself: is there something important or worthwhile exploring in other pathogenesis scenarios than those already favoured by you that may help you improve your own study design, next time you have a fresh look at vitiligo? Vitiligo provides a superb model for the study of many fundamental problems in skin biology and pathology. Therefore, even if it later turns out that, as far as your own vitiligo pathogenesis concept is concerned, you have barked-up the wrong tree most of the time, chances are that you shall anyway have generated priceless new insights into skin function along the way.

Melatonin maintains mitochondrial membrane potential and attenuates activation of initiator (casp-9) and effector caspases (casp-3/casp-7) and PARP in UVR-exposed HaCaT keratinocytes.

Melatonin is a recognized antioxidant with high potential as a protective agent in many conditions related to oxidative stress such as neurodegenerative diseases, ischemia/reperfusion syndromes, sepsis and aging. These processes may be favorably affected by melatonin through its radical scavenging properties and/or antiapoptotic activity. Also, there is increasing evidence that these effects of melatonin could be relevant in keratinocytes, the main cell population of the skin where it would contribute to protection against damage induced by ultraviolet radiation (UVR). We therefore investigated the kinetics of UVR-induced apoptosis in cultured keratinocytes characterizing the morphological and mitochondrial changes, the caspases-dependent apoptotic pathways and involvement of poly(ADP-ribose) polymerase (PARP) activation as well as the protective effects of melatonin. When irradiated with UVB radiation (50 mJ/cm(2)), melatonin treated, cultured keratinocytes were more confluent, showed less cell blebbing, more uniform shape and less nuclear condensation as compared to irradiated, nonmelatonin-treated controls. Preincubation with melatonin also led to normalization of the decreased UVR-induced mitochondrial membrane potential. These melatonin effects were followed by suppression of the activation of mitochondrial pathway-related initiator caspase 9 (casp-9), but not of death receptor-dependent casp-8 between 24 and 48 hr after UVR exposure. Melatonin down-regulated effector caspases (casp-3/casp-7) at 24-48 hr post-UV irradiation and reduced PARP activation at 24 hr. Thus, melatonin is particularly active in UV-irradiated keratinocytes maintaining the mitochondrial membrane potential, inhibiting the consecutive activation of the intrinsic apoptotic pathway and reducing PARP activation. In conclusion, these data provide detailed evidence for specific antiapoptotic mechanisms of melatonin in UVR-induced damage of human keratinocytes.

What are subcutaneous adipocytes really good for?

Our acute awareness of the cosmetic, psychosocial and sexual importance of subcutaneous adipose tissue contrasts dramatically with how poorly we have understood the biology of this massive, enigmatic, often ignored and much-abused skin compartment. Therefore, it is timely to recall the exciting, steadily growing, yet underappreciated body of evidence that subcutaneous adipocytes are so much more than just 'fat guys', hanging around passively to conspire, at most, against your desperate attempts to maintain ideal weight. Although the subcutis, quantitatively, tends to represent the dominant architectural component of human skin, conventional wisdom confines its biological key functions to those of energy storage, physical buffer, thermoregulation and thermoinsulation. However, already the distribution of human superficial adipose tissue, by itself, questions how justified the popular belief is that 'skin fat' (which actually may be more diverse than often assumed) serves primarily thermoinsulatory purposes. And although the metabolic complications of obesity are well appreciated, our understanding of how exactly subcutaneous adipocytes contribute to extracutaneous disease - and even influence important immune and brain functions! - is far from complete. The increasing insights recently won into subcutaneous adipose tissue as a cytokine depot that regulates innate immunity and cell growth exemplarily serve to illustrate the vast open research expanses that remain to be fully explored in the subcutis. The following public debate carries you from the evolutionary origins and the key functional purposes of adipose tissue, via adipose-derived stem cells and adipokines straight to the neuroendocrine, immunomodulatory and central nervous effects of signals that originate in the subcutis - perhaps, the most underestimated tissue of the human body. The editors are confident that, at the end, you shall agree: No basic scientist and no doctor with a serious interest in skin, and hardly anyone else in the life sciences, can afford to ignore the subcutaneous adipocyte - beyond its ample impact on beauty, benessence and body mass.

Oncostatic effects of the indole melatonin and expression of its cytosolic and nuclear receptors in cultured human melanoma cell lines.

Melatonin has been shown to have oncostatic effects on malignant melanoma in vitro and in vivo. We studied the growth suppressive effects of melatonin over a wide range of concentrations in four melanoma cell lines (SBCE2, WM-98, WM-164 and SKMEL-188) representative for different growth stages and phenotype. Melanoma cells were incubated with melatonin 10(-12)-10(-3) M, and proliferation and clonogenicity was assessed at 12 h and 14 days, respectively. We also determined the expression of cytosolic quinone oxidoreductases NQO1, NQO2 (known as MT3 receptor) and nuclear receptor RORalpha by RT-PCR. Melatonin at pharmacological concentrations (10(-3)-10(-7) M) suppressed proliferation in all melanoma cell lines. In SKMEL-188 cells cultured in serum-free media, melatonin at low concentrations (10(-12)-10(-10) M) also slightly attenuated the proliferation. The effects of pharmacological doses of melatonin were confirmed in the clonogenic assay. Expression of NQO1 was detected in all cell lines, whereas NQO2 and nuclear receptor RORalpha including its isoform RORalpha4 were present only in SBCE2, WM-164 and WM-98. Thus, melatonin differentially suppressed proliferation in melanoma cell lines of different behaviour. The intensity of the oncostatic response to melatonin could be related to the cell-line specific pattern of melatonin cellular receptors and cytosolic binding protein expression.

CRH functions as a growth factor/cytokine in the skin.

We tested the effect of CRH and related peptides in a large panel of human skin cells for growth factor/cytokine activities. In skin cells CRH action is mediated by CRH-R1, a subject to posttranslational modification with expression of alternatively spliced isoforms. Activation of CRH-R1 induced generation of both cAMP and IP3 in the majority of epidermal and dermal cells (except for normal keratinocytes and one melanoma line), indicating cell type-dependent coupling to signal transduction pathways. Phenotypic effects on cell proliferation were however dependent on both cell type and nutrition conditions. Specifically, CRH stimulated dermal fibroblasts proliferation, by increasing transition from G1/0 to the S phase, while in keratinocytes CRH inhibited cell proliferation. In normal and immortalized melanocytes CRH effect showed dichotomy and thus, it inhibited melanocyte proliferation in serum-containing medium CRH through G2 arrest, while serum free media led instead to CRH enhanced DNA synthesis (through increased transition from G1/G0 to S phase and decreased subG1 signal, indicating DNA degradation). CRH also induced inhibition of early and late apoptosis in the same cells, demonstrated by analysis with the annexin V stains. Thus, CRH acts on epidermal melanocytes as a survival factor under the stress of starvation (anti-apoptotic) as well as inhibitor of growth factors induced cell proliferation. In conclusion, CRH and related peptides can couple CRH-R1 to any of diverse signal transduction pathways; they also regulate cell viability and proliferation in cell type and growth condition-dependent manners.

Melatonin increases survival of HaCaT keratinocytes by suppressing UV-induced apoptosis.

Melatonin is a potent antioxidant and direct radical scavenger. As keratinocytes represent the major population in the skin and UV light causes damage to these cells, the possible protective effects of melatonin against UV-induced cell damage in HaCaT keratinocytes were investigated in vitro. Cells were preincubated with melatonin at graded concentrations from 10(-9) to 10(-3) m for 30 min prior to UV irradiation at doses of 25 and 50 mJ/cm2. Biological markers of cellular viability such as DNA synthesis and colony-forming efficiency as well as molecular markers of apoptosis were measured. DNA synthesis was determined by [3H]-thymidine incorporation into insoluble cellular fraction, clonogenicity through plating efficiency experiments and apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. DNA synthesis experiments showed a strong protective effect by preincubation with melatonin at concentrations of 10(-4) m (P < 0.01) and 10(-3) m (P < 0.001). Additional postirradiation treatment with melatonin showed no increase in the pre-UV incubation protective effect. These results indicate that preincubation is a requirement for melatonin to exert its protective effects. The mechanism of melatonin's protective effect (10(-6) to 10(-3) m) includes inhibition of apoptosis as measured by TUNEL assay. Moreover, the biological significance of these effects is supported by clonogenic studies showing a significantly higher number of colonies in cultures treated with melatonin compared to controls. Thus, pretreatment with melatonin led to strong protection against UVB-induced damage in keratinocytes.