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Biomedical subjects

B A Gilchrest

Publications and source records attributed to B A Gilchrest.

At least 37 records · Page 2Linked to original sources
PubMed2000

Role of cytoplasmic dynein in melanosome transport in human melanocytes.

Cytoplasmic dynein is a microtubule-associated retrograde-directed motor molecule for transport of membrane-bound organelles. To determine whether cytoplasmic dynein is expressed in melanocytes, we performed reverse transcriptase polymerase chain reaction using melanocyte cDNA and primers complementary to human brain cytoplasmic dynein heavy chain. A polymerase chain reaction product of the expected molecular size was generated and the identity was confirmed by sequence analysis. Western blotting of total melanocyte proteins reacted with an anti-intermediate chain cytoplasmic dynein antibody identified the appropriate 74 kDa band. To determine whether cytoplasmic dynein plays a role in melanosome transport, duplicate cultures were treated with cytoplasmic dynein antisense or sense (control) oligodeoxynucleotides and the cells were observed by high-resolution time-lapse microscopy, which allows visualization of melanosomal aggregates and individual melanosomes. Antisense-treated melanocytes demonstrated a strong anterograde transport of melanosomes from the cell body into the dendrites, whereas melanosome distribution was not affected in sense-treated melanocytes. To determine whether ultraviolet irradiation modifies cytoplasmic dynein expression, melanocyte cultures were exposed to increasing doses of solar-simulated irradiation, equivalent to a mild to moderate sunburn exposure for intact skin. Within 24 h, doses of 5 and 10 mJ per cm2 induced cytoplasmic dynein protein, whereas doses of 30 mJ per cm2 or more were associated with decreased levels of cytoplasmic dynein compared with sham-irradiated controls. Our data show that cytoplasmic dynein participates in retrograde melanosomal transport in human melanocytes and suggest that the altered melanosomal distribution in skin after sun exposure is due, at least in part, to decreased cytoplasmic dynein levels resulting in augmented anterograde transport.

Base Sequence↗
PubMed2000

Mechanisms and implications of the age-associated decrease in DNA repair capacity.

Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6-4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6-4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.

Adult↗
PubMed1999

Protein kinase C-beta activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain.

We have previously shown that protein kinase C-beta (PKC-beta) is required for activation of tyrosinase (Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993) J. Biol. Chem. 268, 11742-11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-beta into nonpigmented human melanoma cells lacking PKC-beta lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-beta in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-beta but not PKC-alpha was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-beta with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-beta. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-beta and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-beta. We conclude that PKC-beta activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.

Animals↗
PubMed1999

Enhanced repair of benzo(a)pyrene-induced DNA damage in human cells treated with thymidine dinucleotides.

The small DNA fragment thymidine dinucleotide (pTpT) stimulates photoprotective responses in mammalian cells and intact skin. These responses include increased melanogenesis (tanning) and enhanced repair of DNA damage induced by ultraviolet (UV) light. Here we show that pTpT treatment of human keratinocytes enhances their repair of DNA damaged by the chemical carcinogen benzo(a)pyrene (BP), as determined by increased expression of a transfected BP-damaged reporter plasmid containing the chloramphenicol acetyltransferase (CAT) gene. The pTpT-enhanced repair of this BP-damaged plasmid is accomplished at least in part through activation of the p53 tumor suppressor protein and transcription factor, because p53-null H1299 cells showed enhanced repair only if previously transfected with a p53-expression vector. To elucidate the mechanism of this enhanced DNA repair, we examined the expression of p21 and proliferating cell nuclear antigen (PCNA), proteins known to be regulated by p53, as well as the XPA protein, which is mutated in the inherited repair-deficient disorder xeroderma pigmentosum (XP) group A and is necessary for the recognition of UV-induced DNA photoproducts. The p53, PCNA and XPA proteins were all up-regulated within 48 h after the addition of pTpT. Taken together, these data demonstrate that pTpT-enhanced repair of DNA damaged by either UV irradiation or chemical mutagens can be achieved in human cells by exposure to small DNA fragments at least in part through the activation of p53 and increased expression of p53-regulated genes.

Benzo(a)pyrene↗
PubMed1999

DNA photodamage stimulates melanogenesis and other photoprotective responses.

Ultraviolet (UV) irradiation is a major source of environmental damage to skin. Melanin pigmentation protects against this damage by absorbing UV photons and UV-generated free radicals before they can react with DNA and other critical cellular components; and UV-induced melanogenesis or tanning is widely recognized as exposed skin's major defense against further UV damage. This article reviews extensive data suggesting DNA damage or DNA repair intermediates directly triggers tanning and other photoprotective responses. Evidence includes the observations that tanning is enhanced in cultured pigment cells by accelerating repair of UV-induced cyclobutane pyrimidine dimers or by treating the cells with UV-mimetic DNA-damaging chemicals. Moreover, small single stranded DNA fragments such as thymidine dinucleotides (pTpT), the substrate for almost all DNA photoproducts, also stimulates tanning when added to cultured pigment cells or applied topically to intact skin. In bacteria, single stranded DNA generated by DNA damage or its repair activates a protease that in turn derepresses over 20 genes whose protein products enhance DNA repair and otherwise promote cell survival, a phenomenon termed the SOS response. Interestingly, pTpT also enhances repair of UV-induced DNA damage in human cells and animal skin, at least in part by activating the tumor suppressor protein and transcription factor p53 and thus upregulating a variety of gene products involved in DNA repair and cell cycle regulation. Together, these data suggest that human cells have an evolutionarily conserved SOS-like response in which UV-induced DNA damage serves as signal to induce photoprotective responses such as tanning and increased DNA repair capacity. The responses can also be triggered in the absence of DNA damage by addition of small single-stranded DNA fragments such as pTpT.

Animals↗
PubMed1999

Thymidine dinucleotide mimics the effect of solar simulated irradiation on p53 and p53-regulated proteins.

The tumor suppressor protein p53 participates in DNA repair and cell cycle regulation in response to injuries like ultraviolet (UV) irradiation. We have previously reported that the thymidine dinucleotide (pTpT), a common target for DNA photoproduct formation by UV light, mimics many effects of UV irradiation in cultured skin-derived cells, at least in part through the activation of p53. In this report we compare the effects of solar-simulated irradiation and pTpT on p53 and p53-regulated proteins involved in cellular growth arrest and DNA repair in cultured human dermal fibroblasts. We find that, like UV irradiation, pTpT increases the levels of p53, p21, and proliferating-cell nuclear antigen. The magnitude and time course of the inductions are UV dose dependent and consistent with known regulatory interactions among these nuclear proteins. These data confirm and expand previous studies of UV effects on nuclear proteins involved in cell cycle regulation and DNA repair. Our observations suggest that such protective effects can also be induced by pTpT in the absence of initial DNA damage, rendering cells more capable of responding to subsequent DNA damage.

Cells, Cultured↗
PubMed1999

Signaling pathways mediating melanogenesis.

Pigmentation of the skin, due to the synthesis and dispersion of melanin in the epidermis, is of great cosmetic and societal significance. It is also the key physiologic defense against sun-induced injuries such as sunburn, photocarcinogenesis and photoaging. During recent decades, there has been a dramatic increase in skin cancers, including melanoma, due to habitual sun exposure (Rigel, 1992; Weinstock, 1989). At present, in the United States, about one in 75 individuals is projected to develop malignant melanoma during his or her lifetime (Rigel, 1992). Unfortunately, progress in preventing sun-related injuries has been slow, in part due to lack of understanding of the molecular mechanisms involved in pigmentation. This article reviews recent progress in identifying signal transduction pathways that mediate melanogenesis.

Animals↗
PubMed1998

Activation of cAMP-dependent protein kinase is required for optimal alpha-melanocyte-stimulating hormone-induced pigmentation.

The cAMP-dependent pathway has been long presumed to play a critical role in mediating alpha-melanocyte-stimulating hormone (alpha-MSH)-induced pigmentation, but it has never been demonstrated that this pathway is obligatory. In order to determine whether the cAMP-dependent pathway is required for a alpha-MSH-induced pigmentation, we inhibited the activity of cAMP-dependent protein kinase (PKA), the main kinase mediating in this pathway, by introducing a physiologic cAMP-dependent protein kinase inhibitor (PKI) into S91 murine melanoma cells and then measuring pigment response after alpha-MSH stimulation. Cells were stably transfected either with the pMXX-PKI expression vector that encodes the active part of PKI (the amino terminal 1-31 amino acids) under a metallothionein-inducible promoter and the pSV2-Neo expression vector alone. As expected, treatment of transfected cells with 1 microM CdCl2 for 24 h induced the expression of PKI mRNA in cells transfected with both vectors, but not in cells transfected with the pSV2-Neo expression vector alone. Subsequent treatment of these transfected cells with alpha-MSH for 5-6 days in the continual presence of 1 microM CdCl2 resulted in inhibition of PKA activity by 30-40% in cells expressing PKI. Parallel measurements revealed that alpha-MSH-increased melanin content five- to six-fold in control cells transfected with pSV2-Neo alone, while there was only a two-fold increase in PKI-expressing cells, a 40-50% inhibition in alpha-MSH-induced total melanin content. alpha-MSH-induced tyrosinase activity and tyrosinase mRNA and protein levels measured in parallel were also inhibited by 40-50% in PKI-expressing cells compared to control cells transfected with pSV2-Neo alone. Together, these results demonstrate for the first time that activation of PKA through the cAMP-dependent pathway is required for optimal alpha-MSH-induced pigmentation.

Animals↗
PubMed1998

Protein kinase C-alpha levels are inversely associated with growth rate in cultured human dermal fibroblasts.

Human dermal fibroblasts are known to express the alpha, delta, epsilon, and zeta isoforms of protein kinase C (PKC). We asked whether the growth of human dermal fibroblasts correlates with expression of a particular PKC isoform. Of total PKC activity measured in the presence of calcium, a condition permissive for activation of all PKC isoforms, 75%) was contributed by PKC-alpha, suggesting that PKC-alpha is the dominant isoform in human dermal fibroblasts. We then further studied PKC-alpha under different culture conditions and in cultures derived from different aged donors. In both subconfluent and confluent cultures, total PKC activity and the level of PKC-alpha protein were consistently higher in slowly proliferating adult cells than in more rapidly proliferating newborn cells. Moreover, in newborn fibroblasts density strongly influenced these parameters. At subconfluent density, when cells were dividing exponentially, total PKC activity was 345+/-63 cpm/,ug protein; whereas at confluent density, when cells were growth arrested, it was 6-7 fold higher, 2334+/-50 cpm/ug protein. Immunoblot analysis using a specific monoclonal antibody against PKC-alpha exhibited a similar 6-7 fold increase in the level of PKC-alpha protein at confluent density. However, in adult cells, density had no influence on the already high total activity or level of PKC-alpha. To further determine whether the increases in the levels of total PKC activity and the alpha isoform correlate with the decreased growth rate, a characteristic of both adult donor-derived and confluent cells, total PKC activity and the level of PKC-alpha in subconfluent quiescent cells was compared to that in paired exponentially growing cells at the same density. Total PKC activity was 8836+/-71 cpm/microg protein in subconfluent quiescent cells versus 4415+/-175 cpm/microg protein in dividing cells. The level of PKC-alpha protein was also 2-3 fold higher in quiescent than in growing cultures. However, the amount of PKC-alpha mRNA in these two conditions was identical as determined by northern blot analysis. Taken together, these results suggest an inverse relationship between the levels of total PKC activity and PKC-alpha protein and fibroblast growth rate that is regulated at the post-transcriptional level.

Adult↗
PubMed1998

Post-transcriptional regulation of UV induced TNF-alpha expression.

Ultraviolet (UV) irradiation exerts multiple effects on skin cells, including the induction of several cytokines involved in immunomodulation. Specifically, UV irradiation has been shown to upregulate the level of tumor necrosis factor-alpha (TNF-alpha) mRNA in keratinocytes. To determine whether the induction of TNF-alpha mRNA is regulated by transcriptional or post-transcriptional mechanisms, we examined cells of keratinocytic lineage (SCC12F) for steady state level, transcription rate, and stability of TNF-alpha mRNA after UV irradiation. Within 4 h there was a 20-40-fold induction of TNF-alpha mRNA that persisted at lower levels through 48 h. Consistently, TNF-alpha protein secretion increased at 24 and 48 h after UV irradiation. UV irradiation increased the half-life of TNF-alpha mRNA from approximately 35 min to approximately 10 h. Conversely, the transcription rate of the TNF-alpha gene increased < 2-fold at the time of peak mRNA steady state levels. Thus, post-transcriptional mechanisms play a major role in UV induced TNF-alpha transcript level.

Cell Line↗
PubMed1998

What controls melanogenesis?

The pigments eumelanin and pheomelanin are the visually most striking products of specialized neural crest-derived cells (melanocytes), and provide color to both epidermis and hair shafts. While the intriguing and controversial biological functions of these multifaceted heteropolymers will be discussed in a later feature, here it is explored how their generation (melanogenesis) is controlled. For decades, this has been the object of much controversy, the salient features of which are delineated in the following contributions.

Animals↗
PubMed1998

Keratinocytes and dermal factors activate CRABP-I in melanocytes.

Recognition that cellular retinoic acid binding protein (CRABP)-I and CRABP-II are found in different cell types has provided additional support for the presumably divergent roles of these two proteins in mediating retinoic acid (RA) effects in human skin. CRABP-II is expressed in fibroblasts and keratinocytes, and CRABP-I in as yet unidentified cells, possibly epidermal melanocytes. Recently, we demonstrated that each of these RA-binding proteins in human skin possesses two classes of binding sites, possibly related to the state of phosphorylation of the proteins. We now characterize the cutaneous origin of CRABP-I further using an anion-exchange HPLC assay that allows effective separation of the two proteins in human skin, and a fluorescent in situ hybridization technique. We report that CRABP-I is expressed in isolated melanocytes at the mRNA level, although under these circumstances the protein has minimal RA-binding activity, and that keratinocytic and dermal influences are required for CRABP-I activity in melanocytes. This melanocyte origin for CRABP-I and the improvement by RA of the irregular hyperpigmentation associated with photoaging led us to examine the effects of RA using various cellular associations, from conventional pure cultures of melanocytes grown on plastic dishes to a pigmented skin equivalent consisting of melanocytes and keratinocytes grown on a dermal equivalent. We established that the inhibitory effects of RA on melanogenesis do not result from a direct effect on melanocytes alone but also involve keratinocytes and dermal influence. These data expand our understanding of cell-to-cell signaling in cutaneous pigmentation, and strongly suggest a role for CRABP-I in mediating RA effects on melanogenesis.

Cell Communication↗
PubMed1998

Aging versus photoaging: postulated mechanisms and effectors.

The differences between intrinsic aging and photoaging are reviewed. The various model systems currently employed for the studies of aging and photoaging are discussed. Findings on age associated decrements in receptor/ligand mediated signaling as well as changes during cellular senescence in the expression of nuclear transcription factors are described. The role of telomere shortening and oxidative damage in the aging process is explained. At the cellular level, genetic and behavioral differences between aging and photoaging are illustrated with particular emphasis on changes in the structure and function of the tumor suppressor gene p53.

Aging↗
PubMed1997

Enhancement of DNA repair in human skin cells by thymidine dinucleotides: evidence for a p53-mediated mammalian SOS response.

Thymidine dinucleotide (pTpT) stimulates melanogenesis in mammalian pigment cells and intact skin, mimicking the effects of UV irradiation and UV-mimetic DNA damage. Here it is shown that, in addition to tanning, pTpT induces a second photoprotective response, enhanced repair of UV-induced DNA damage. This enhanced repair results in a 2-fold increase in expression of a UV-damaged chloramphenicol acetyltransferase expression vector transfected into pTpT-treated skin fibroblasts and keratinocytes, compared with diluent-treated cells. Direct measurement of thymine dimers and (6-4) photoproducts by immunoassay demonstrates faster repair of both of these UV-induced photoproducts in pTpT-treated fibroblasts. This enhanced repair capacity also improves cell survival and colony-forming ability after irradiation. These effects of pTpT are accomplished, at least in part, by the up-regulation of a set of genes involved in DNA repair (ERCC3 and GADD45) and cell cycle inhibition (SDI1). At least two of these genes (GADD45 and SDI1) are known to be transcriptionally regulated by the p53 tumor suppressor protein. Here we show that pTpT activates p53, leading to nuclear accumulation of this protein, and also increases the specific binding of this transcription factor to its DNA consensus sequence.

Cells, Cultured↗
PubMed1997

Binding of beta-amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of aggregated beta-amyloid peptide, presumed to play a pathogenic role, and by preferential loss of neurons that express the 75-kD neurotrophin receptor (p75NTR). Using rat cortical neurons and NIH-3T3 cell line engineered to stably express p75NTR, we find that the beta-amyloid peptide specifically binds the p75NTR. Furthermore, 3T3 cells expressing p75NTR, but not wild-type control cells lacking the receptor, undergo apoptosis in the presence of aggregated beta-amyloid. Normal neural crest-derived melanocytes that express physiologic levels of p75NTR undergo apoptosis in the presence of aggregated beta-amyloid, but not in the presence of control peptide synthesized in reverse. These data imply that neuronal death in Alzheimer's disease is mediated, at least in part, by the interaction of beta-amyloid with p75NTR, and suggest new targets for therapeutic intervention.

3T3 Cells↗
PubMed1997

Fas/Fas ligand interaction contributes to UV-induced apoptosis in human keratinocytes.

Keratinocytes in human skin undergo apoptosis during various inflammatory processes and after ultraviolet (UV) irradiation. To determine if keratinocyte apoptosis may be mediated by the Fas/APO-1 receptor (CD95), a signal transduction pathway known to initiate programmed cell death of lymphocytes, we investigated Fas expression, modulation, and function in keratinocytes. Keratinocytes constitutively expressed the 2.5- and 1.9-kb Fas transcripts, as well as the 43-kDa Fas protein. Treatment of interferon-gamma-stimulated keratinocytes with Fas agonistic antibody significantly promoted their cell death, indicating that Fas in keratinocytes is functional. UV irradiation induced Fas mRNA expression within 16 to 24 h and Fas protein within 24 h and through 48 h after irradiation. Furthermore, keratinocytes constitutively expressed Fas ligand (FasL) mRNA and protein. UV irradiation induced FasL mRNA as early as 4 h after irradiation and elevated FasL mRNA levels were maintained for at least 24 h postirradiation. Moreover, a FasL neutralizing antibody significantly reduced UV-induced apoptosis of IFN-gamma-treated keratinocytes. Our data strongly suggest that the Fas system contributes to keratinocyte apoptosis in UV-irradiated human skin.

Antibodies, Monoclonal↗