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Biomedical subjects

B A Gilchrest

Publications and source records attributed to B A Gilchrest.

At least 91 records · Page 5Linked to original sources
PubMed1994

Retinoids and the skin.

Retinoids are a group of naturally occurring and synthetic compounds with vitamin A-like biological activity. They play an important role in vision, reproduction, growth, and epithelial cell differentiation. Recent discoveries of specific retinoid cellular binding proteins and nuclear receptors have led to a better (although not complete) understanding of the complex mechanisms of retinoid action. Numerous clinical studies have demonstrated beneficial effects of retinoids on skin diseases such as acne, psoriasis, ichthyoses, keratodermas, skin cancers and their precursors, as well as a reversal of the effects of photoaging.

Animals↗
PubMed1994

The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.

We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors.

Amino Acid Sequence↗
PubMed1994

Role of integrins in melanocyte attachment and dendricity.

Integrins are a family of proteins known to mediate attachment of cells to extracellular matrix materials. The substratum specificity and cation dependence of specific integrin heterodimers have been extensively characterized, and to a lesser degree specialized roles in cell attachment versus dendricity have been defined in some cell types. In the past decade, melanocyte attachment rate and morphology have been found to have strong substratum dependence, suggesting a major role for integrins in these processes. In order to investigate this aspect of pigment cell biology, human newborn melanocytes were subjected to flow cytometry analysis and plated on a variety of substrata under conditions known to promote or block the binding of specific integrin pairs. Melanocyte attachment to laminin and type IV collagen was promoted by Mg2+ and Mn2+ but not by Ca2+, in the range of concentrations examined. However, dendrite outgrowth from melanocytes already attached on laminin or type IV collagen was promoted by Ca2+ to a far greater degree than by Mg2+, and Mn2+ had no effect on dendrite outgrowth. Flow cytometry analysis revealed that melanocytes expressed beta 1, alpha 2, alpha 3, alpha 5, alpha 6 and alpha v integrin subunits as well as the alpha v beta 3 heterodimer. The influence of substratum on the profile of integrin expression was minimal, but alpha 6 and beta 1 integrins were observed by confocal microscopy to be expressed over the entire cell surface, while alpha 2, alpha 5 and alpha v beta 3 integrins localized along dendritic processes or at their tips. In accordance with the implications of these distribution patterns, anti-beta 1 and anti-alpha 6 integrin monoclonal antibodies blocked melanocyte attachment to laminin, while anti-alpha 2, anti-alpha 5 and anti-alpha v beta 3 inhibited dendrite outgrowth but did not block substratum attachment on either laminin or type IV collagen. On the basis of these data and the known characteristics of integrin molecules, we conclude that melanocyte attachment to laminin is mediated primarily by alpha 6 beta 1 integrin in a Ca(2+)-independent, Mg(2+)- and/or Mn(2+)-dependent manner, while dendrite outgrowth on laminin and type IV collagen requires extracellular Ca2+ and is mediated by alpha v beta 3 as well as alpha 2 and alpha 5 integrins.

Antibodies, Monoclonal↗
PubMed1994

E-cadherin is the major mediator of human melanocyte adhesion to keratinocytes in vitro.

E- and P-cadherin are calcium (Ca2+)-dependent cell adhesion molecules important in the morphogenesis and maintenance of skin structure. By use of flow cytometry and specific antibodies, we now show that cultured human melanocytes express E- and P-cadherin on their surfaces, and that these molecules have the same characteristics as reported for other cell types. Specifically, melanocyte cadherins are sensitive to trypsin digestion in the absence of Ca2+ and are protected from trypsin degradation by Ca2+, and are functional at 37 degrees C but not at 4 degrees C. We further show that melanocytes contain mRNA transcripts encoding both E- and P-cadherin. Adhesion of cultured melanocytes to keratinocyte monolayers is abolished by pre-treatment of the melanocytes with trypsin/EDTA, which degrades E- and P-cadherins, is greatly reduced by anti-E-cadherin antibodies and is slightly reduced by antibodies to P-cadherin, alpha 2, alpha 3 and beta 1 integrins. In contrast to normal melanocytes, eight of nine melanoma cell lines lacked E-cadherin (or expressed markedly reduced levels) and five were negative for P-cadherin. Melanoma cells also failed to adhere to keratinocyte monolayers. These results demonstrate that normal human melanocytes express functional E- and P-cadherin and that E-cadherin is primarily responsible for adhesion of human melanocytes to keratinocytes in vitro. In addition, transformed melanocytes express markedly reduced levels of E- and P-cadherin, and exhibit decreased affinity for normal keratinocytes in vitro, suggesting that loss of cadherins may play a role in melanoma metastasis.

Base Sequence↗
PubMed1994

Evaluation of the photoprotective effect of oral vitamin E supplementation.

BACKGROUND AND DESIGN: Solar-induced cutaneous damage is mediated partly via oxidative pathways. Some evidence exists for a photoprotective role of antioxidants. In a double-blind, placebo-controlled study, we examined the effect of a long-term, orally administered antioxidant against UV-induced epidermal damage. Healthy human subjects supplemented their usual diet daily with either 400 IU of oral vitamin E (alpha-tocopherol acetate) or placebo over a 6-month period. Minimal erythema dose and histologic response to threefold minimal erythema dose exposure were determined at baseline, 1 month, and 6 months. RESULTS: The minimal erythema dose did not vary substantially at the three time points within each treatment group or in the vitamin E-supplemented group vs the placebo group. The number of sunburn cells produced by a threefold minimal erythema dose exposure was also not significantly different between the two groups. Of note, however, vitamin E levels in plasma increased only modestly and in skin biopsy specimens were unchanged following 1 month and 6 months of supplementation. CONCLUSIONS: No clinical or histologic difference in the response to UVB could be detected between the placebo and vitamin E-supplemented groups. In this small study, daily ingestion of 400 IU of oral alpha-tocopherol daily does not provide meaningful photoprotection.

Administration, Oral↗
PubMed1994

Aging and photoaging affect gene expression in cultured human keratinocytes.

BACKGROUND: Aging has two components: changes that occur in all individuals with the passage of time alone and changes that occur to varying degrees in different individuals as a result of repeated environmental injury. In the skin, the major environmental influence is sun exposure, and the combined effect of aging and sun damage has been termed photoaging. To determine whether aging and photoaging have characteristic effects on gene expression, we performed Northern blot analysis of several genes in early-passage cultured keratinocytes derived from donors of different ages (newborn, young adult, and old adult) and from paired sun-exposed (photoaged) and sun-protected sites of old-adult donors. OBSERVATIONS: We examined genes involved in cell division, immunomodulation, or differentiation. Aging alone strikingly increased the baseline expression of the differentiation-associated SPR2 and interleukin 1 receptor antagonist (IL-1ra) genes but had relatively little effect on the UV inducibility of any genes studied. In contrast, photoaging markedly increased the inducibility of the c-fos proto-oncogene and decreased the baseline expression of SPR2 and IL-1ra relative to that in cells derived from sun-protected skin of the same donors. CONCLUSIONS: Both aging and photoaging alter the expression of selected genes that are implicated in growth, differentiation, immunomodulation, and UV response in human epidermis. This may explain, in part, the predisposition to photocarcinogenesis in chronically sun-exposed skin of older individuals.

Adult↗
PubMed1993

The beta isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells.

We have investigated the role of protein kinase C (PKC) in human melanogenesis. The level of PKC activity paralleled the total melanin content in cultured newborn melanocytes. Activation of PKC by treatment with 5 x 10(-7) M phorbol dibutyrate acutely caused a doubling in the activity of tyrosinase, the rate-limiting enzyme in melanogenesis, known to correlate directly with melanin synthesis in these cells. When PKC was depleted to 5-10% of initial levels, there was a 40-50% parallel reduction in tyrosinase activity; and regeneration of PKC activity was associated with the recovery of tyrosinase activity. By Northern blot analysis, the alpha and beta but not the gamma isoforms were detectable in melanocytes. By Western blot analysis, the racially determined pigment level in cultured melanocytes correlated with PKC-beta protein expression. In a pigmented human melanoma line (P-MM4, 20-30 ng melanin/micrograms protein)and its nonpigmented subclone (NP-MM4, undetectable melanin), PKC-alpha mRNA was expressed in both, whereas PKC-beta mRNA was detectable only in P-MM4 cells. Tyrosinase protein level was comparable in both cell lines. When NP-MM4 cell lysate was incubated with melanocyte lysate known to contain PKC-beta, tyrosinase activity per microgram of melanocyte protein in the combined lysate increased, consistent with activation of the previously inactive tyrosinase of NP-MM4 origin. Moreover, NP-MM4 cells transiently transfected with PKC-beta cDNA increased tyrosinase activity from undetectable to detectable levels. These combined data show that PKC-beta regulates human melanogenesis by activating tyrosinase.

Blotting, Northern↗
PubMed1993

Gene modulation accompanying differentiation of normal versus malignant keratinocytes.

Using tetradecanoylphorbol 13-acetate (TPA), a known inducer of epithelial cell differentiation, and Northern blot analysis, we investigated in normal versus well-differentiated malignant keratinocytes the modulation of genes implicated in their growth or differentiated function. In normal keratinocytes transient c-fos induction was detected within 30 min after stimulation and was followed by rapid down regulation of the proto-oncogenes c-myc and epidermal growth factor receptor. Within hours after stimulation mRNA levels for three well-characterized differentiated keratinocyte products, involucrin, interleukin-1 beta (IL1-beta), and fibronectin, were induced, as were those for a growth arrest and DNA damage-inducible (GADD 153) gene and a small proline-rich (SPR1) gene, both known to be associated with differentiation but with of as yet unknown functions. Heat shock protein 70 gene was initially down regulated and was induced only after 48 h. The well-differentiated malignant keratinocyte cell line differed in that the c-fos, GADD, SPR1, and IL1-beta genes had several-fold higher induction, but involucrin mRNA was undetectable and fibronectin mRNA was only minimally induced after TPA stimulation. Malignant cells reached terminal differentiation faster than normal keratinocytes as measured by inability to exclude trypan blue dye, and in situ hybridization using a riboprobe for the differentiation-associated SPR1 gene showed that normal keratinocytes constitutively express this transcript while malignant keratinocytes with virtually identical morphology and growth rate do not. These studies greatly expand our understanding of gene activation and down regulation during normal keratinocyte differentiation and imply that a malignant cell line, even when retaining the phenotype of normal cells, differs in its response to outside stimuli, furnishing at best an imperfect model for investigating the molecular mechanisms of cellular differentiation.

CCAAT-Enhancer-Binding Proteins↗
PubMed1993

Effect of donor age on epidermal growth factor processing in man.

Epidermal growth factor (EGF) is a well-characterized mitogen whose effectiveness decreases with age both in vivo and in vitro. Previous studies utilizing the Hayflick aging model of early versus late-passage fibroblasts failed to demonstrate changes that might account for the unresponsiveness. In contrast, we now report striking differences in EGF receptor (EGFR) number, affinity, and rate of EGF/EGFR internalization in early-passage dermal fibroblasts derived from newborn versus young adult versus old adult donors. These data demonstrate critical differences between the two models of cellular aging, provide an explanation for the age-associated loss of EGF responsiveness, and may explain in part the tendency toward impaired wound healing in the elderly.

Adolescent↗
PubMed1993

Protein kinase C: biochemical characteristics and role in melanocyte biology.

The protein kinase C (PKC) family of proteins, consisting of at least ten isoforms, has been shown to regulate major cellular functions, including the growth and differentiation in many cell types. Use of PKC activators and inhibitors in combination with molecular biology techniques, has permitted detailed exploration of their specific intracellular actions. Recently, studies have implicated PKC specifically in the regulation of growth and differentiated function in melanocytes. In particular, the beta-isoform of PKC was shown to regulate human melanogenesis through activation of tyrosinase, the rate limiting enzyme in melanogenesis. This article reviews the role of PKC in melanocyte biology.

Animals↗
PubMed1993

Treatment of human melanocytes and S91 melanoma cells with the DNA repair enzyme T4 endonuclease V enhances melanogenesis after ultraviolet irradiation.

Tanning is a protective response of ultraviolet (UV)-irradiated skin that decreases damage from subsequent sun exposures by increasing the epidermal content of melanin, a brown-black pigment that absorbs light energy throughout the UV and visible portions of the electromagnetic spectrum. The melanin pigment is made by epidermal melanocytes and transferred to surrounding keratinocytes. The action spectrum, time course, and histologic features of tanning are well studied, but the initiating molecular events are unknown. Previous work has shown that T4 endonuclease V, a prokaryotic DNA repair enzyme that catalyzes the first and rate-limiting step in repair of UV-induced pyrimidine dimers, delivered in carrier liposomes (T4N5), enhances repair of UV-induced DNA damage in cultured human cells and protects against photocarcinogenesis in an animal model. We now report that T4N5 treatment enhances UV-induced melanogenesis, as measured by melanin content, tyrosinase activity, 14C-dopa incorporation, and visual assessment in both S91 murine melanoma cells and human melanocytes. T4N5 treatment also increases cell yields following UV irradiation. These data suggest that tanning can be stimulated through enhanced DNA repair.

Cell Division↗
PubMed1993

Retinoids and state of differentiation modulate CRABP II gene expression in a skin equivalent.

Cellular retinoic acid-binding proteins (CRABPs) are a family of proteins that specifically bind retinoic acid (RA) and have been implicated in mediating its action, although their exact function is still unknown. Two CRABPs have been identified and cloned. CRABP I is present in many tissues and cultured cells; CRABP II, first detected in embryonic and neonatal skin of rats and chicks, is now recognized as the predominant form in human epidermis. Using a human living skin equivalent model composed of a dermis and an epidermis and human cDNAs recently cloned in our laboratory, we have studied the effects of 10(-6) M RA and etretin (ET) on the expression of CRABPs under different culture conditions intended to favor greater or lesser degrees of epidermal differentiation. Total cellular RNA was isolated separately from the dermis and epidermis and processed for northern blot analysis. At a presumptive physiologic RA concentration, only the gene for CRABP II, and not for CRABP I, was expressed. CRABP II transcripts were far more abundant on a per cell basis in epidermal keratinocytes than in dermal fibroblasts under all conditions studied. Epidermal differentiation, stimulated by air exposure of the cultures, tended to enhance CRABP II expression, and treatment with presumptive therapeutic concentrations of the two retinoid compounds tended to decrease CRABP II expression. Opposite effects of air exposure and retinoid treatment were observed on steady state levels of mRNA for selected markers of epidermal differentiation: involucrin, transglutaminase, and spr I. These results are consistent with earlier work at the protein level examining the effect of retinoids on CRABP activity and state of differentiation both in vivo and in vitro. Thus, the skin equivalent appears to be an excellent model system for investigating the role of CRABPs in mediating retinoid effects at the cellular and molecular levels.

Acitretin↗
PubMed1993

Wound healing and aging.

Age-related differences in wound healing have been clearly documented. Although the elderly can heal most wounds, they have a slower healing process, and all phases of wound healing are affected. The inflammatory response is decreased or delayed, as is the proliferative response. Remodeling occurs, but to a lesser degree, and the collagen formed is qualitatively different. Diseases that affect wound healing are more prevalent in the elderly and have a greater adverse effect on healing than in young adults. Thus, particularly in the elderly, concomitant medical problems should be treated vigorously to allow for maximum healing. Recent trials of novel therapies to enhance wound healing suggest, however, that much can be done to improve the prognosis of elderly patients with risk factors known to adversely affect wound healing.

Adult↗
PubMed1992

Cytoskeletal events underlying dendrite formation by cultured pigment cells.

In contrast to neurite outgrowth, pigment cell dendrite formation is relatively unstudied. Keratinocyte-conditioned medium (KCM) induces a striking dendricity in human melanocytes and B16 melanoma cells that is detectable within 30 min, maximal in 24-48 hr, and quantifiable by computerized image analysis. Cytochalasin B (CB), known to disrupt actin microfilaments, completely blocks dendrite formation if added to cultures before or with KCM. This effect is rapidly reversible, and dendrites appear within 1 hr after refeeding with KCM alone. In contrast, CB treatment fails to disrupt existing dendrites previously induced by KCM. Agents known to cause microtubule disassembly (colchicine, nocodazole, or vinblastine) do not inhibit dendrite formation if added before or with KCM. In contrast, these agents disrupt established dendrites. Inhibition of protein synthesis with cycloheximide or actinomycin D completely blocks dendrite formation, but if cultures are provided fresh KCM lacking protein synthesis inhibitors, dendrites reappear within 24 hr. Actin microfilaments visualized with a monoclonal antibody or rhodamine-phalloidin are poorly organized in untreated cells, but form numerous fibers localized along dendrites in KCM-treated cells. Microtubules visualized with a monoclonal anti-tubulin antibody are localized in the center of dendrites. These cytoskeletal changes occur without altering beta actin or beta tubulin mRNA levels. Taken together, these data implicate actin microfilaments in dendrite outgrowth, but not in maintenance, and conversely microtubules in dendrite maintenance but not in formation. These keratinocyte-induced changes involving beta actin and beta tubulin polymerization appear to require both new protein synthesis and post-translational regulation. The observed similarities between melanocytes and other neural crest-derived cells suggest that cutaneous pigment cells might serve as an alternative model for studies of neurite outgrowth.

Actins↗
PubMed1992

Modulation of mRNA levels during human keratinocyte differentiation.

Cultures of human keratinocytes provide an excellent model system in which to study differentiation. Using the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and calcium, two agents known to induce keratinocyte differentiation in vitro, we examined the expression of the genes encoding c-fos, c-myc, and c-jun; involucrin, a protein precursor of the keratinocyte cornified envelope; and L-7, a ribosomal protein. Overall, at the doses studied, TPA induced a more rapid and profound differentiation than did calcium, as evaluated by culture morphology and northern blot analysis. Our studies showed a constant low level of c-fos and c-jun expression in unstimulated cells with no significant change after addition of either TPA or calcium except when transcript breakdown was inhibited by cycloheximide. The c-myc proto-oncogene, known to have a high constitutive expression in actively proliferating cells, was strongly downregulated by TPA, but calcium had no effect over a 32 hour period, consistent with the greater growth inhibition of TPA in this system. Involucrin was induced about ninefold by both TPA and calcium as early as 8 hours after stimulation, suggesting transcriptional regulation of this gene during differentiation. L-7, recently demonstrated to be downregulated in late passage human fibroblasts in an in vitro model of senescence, was also strongly downregulated by either TPA or calcium, consistent with an interrelationship between the basic cellular processes of aging and differentiation. These finding expand our knowledge of the differentiation process in human keratinocytes.

Calcium↗