Biomedical subjects
M S Eller
Publications and source records attributed to M S Eller.
Epidermal differentiation enhances CRABP II expression in human skin.
The cellular retinoic acid-binding proteins (CRABP I and II) are thought to mediate the effects of retinoic acid on target cells. We have used riboprobes complementary to CRABP I and II mRNAs to study the expression of these messages in normal and abnormal human skin. CRABP II was expressed predominantly in the suprabasal layers of the epidermis, with stronger expression in newborn than in sun-protected adult skin. Interestingly, the epidermis adjacent to or overlying squamous cell or basal cell carcinomas also showed strong expression, whereas the tumor cells were negative, with the exception of more differentiated cells surrounding the "keratin pearls" within squamous cell carcinomas. CRABP II mRNA was also found in the more differentiated cells of the hair follicles, in the outer root sheath. CRABP I message was undetectable in the epidermis or in the dermis of normal skin but was detected in the cells of the papillary dermis surrounding basal and squamous cell carcinomas. These data suggest that increased levels of CRABP II mRNA accompany keratinocyte differentiation in vivo.
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.
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.
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.
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.
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.
The molecular cloning and expression of two CRABP cDNAs from human skin.
Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). We have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABP I cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. Northern blot analysis showed CRABP II mRNA level was only slightly reduced by addition of 10(-6) or 10(-5) M RA to cultures of neonatal foreskin-derived fibroblasts, as was the CRABP I mRNA level in cultured human gut epithelial cells. In contrast, expression of CRABP II mRNA by cultured neonatal keratinocytes was strongly downregulated by RA. We conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. Our data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.
Myosin light chain 3 synthesis during chick pectoralis muscle development in ovo.
The level of myosin light chain 3 (LC3) in vertebrate skeletal muscle is developmentally regulated in a tissue-specific manner. We have used the RNA-cDNA hybridization assay to quantitate LC3 mRNA levels at various stages of chick pectoralis muscle development in ovo. The LC3 mRNA was found significantly in breast muscle only on Day 16 in ovo and later, the level of mRNA ranging from about 30 to 32% of that present in adult tissue. These values are in good agreement with the corresponding levels of LC3 in embryonic muscle. These results do not support the earlier reports that the protein and mRNA for LC3 accumulate in a noncoordinate manner in embryonic pectoralis muscle and they suggest that LC3 synthesis in ovo is regulated primarily at the transcriptional level.
Isolation and characterization of a translation inhibitor from human term placenta.
An inhibitor of protein synthesis has been isolated from free cytoplasmic ribonucleoprotein particles of human term placenta. The inhibitor is resistant to phenol, DNase, proteinase K, and heating at 100 degrees C, but is sensitive to alkaline hydrolysis. These data suggest that the inhibitor is RNA. Experiments provide evidence that this preparation contains no RNase contaminant and does not induce an RNase in this assay system. Three lines of evidence suggest that the inhibitor acts at the initiation of protein synthesis in the wheat germ translation system. First, a lag occurs before cessation of translation when the inhibitor is added to translating polyribosomes. This lag is identical to that seen upon the addition of aurintricarboxylic acid, a known inhibitor of initiation. Second, sucrose gradient analyses demonstrate that, when the inhibitor is present at the start of translation, 40 S complexes form, but neither 80 S complexes nor polyribosomes are seen. Third, gradient analyses show that, when the inhibitor is added to translating polyribosomes, 40 S complexes accumulate with a progressive loss of polyribosomes. Finally, the extent of inhibition depends upon the amount of wheat germ extract added to the reaction mixture and not the amount of mRNA present. This suggests an interaction between the inhibitor and a component of the wheat germ extract.