Biomedical subjects

Hee-Young Park

Publications and source records attributed to Hee-Young Park.

9 recordsLinked to original sources

PubMed2006

Bone morphogenetic protein-4, a novel modulator of melanogenesis.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta family, signal in many cells including neural precursors. Two receptors, types 1 and 2, coordinately mediate BMP signaling, and type 1 receptor has two forms: A and B. Using RT-PCR we found that neural crest-derived human melanocytes express BMP receptor-1A, -1B, and -2. Furthermore, melanocytes and the surrounding keratinocytes express BMP-4, suggesting both autocrine and paracrine effects of this molecule. Moreover, BMP-4 supplementation of cultured human melanocytes decreases melanin synthesis, tyrosinase mRNA, and protein. The mechanism of this BMP-4 effect on tyrosinase and ultimately on melanogenesis involves modest decreases of tyrosinase transcription rate and mRNA stability. Moreover, ultraviolet irradiation, the best recognized environmental stimulator of melanogenesis, down-regulated the mRNA of BMP receptor-1B in melanocytes. Our data provide evidence of a novel regulatory pathway for melanogenesis in human skin.

Apoptosis↗

PubMed2006

MITF mediates cAMP-induced protein kinase C-beta expression in human melanocytes.

The cAMP-dependent pathway up-regulates MITF (microphthalmia-associated transcription factor), important for key melanogenic proteins such as tyrosinase, TRP-1 (tyrosinase-related protein 1) and TRP-2. We asked whether MITF is also a key transcription factor for PKC-beta (protein kinase C-beta), required to phosphorylate otherwise inactive tyrosinase. When paired cultures of human melanocytes were treated with isobutylmethylxanthine, known to increase intracellular cAMP, both protein and mRNA levels of PKC-beta were induced by 24 h. To determine whether MITF modulates PKC-beta expression, paired cultures of human melanocytes were transfected with dn-MITF (dominant-negative MITF) or empty control vector. By immunoblotting, PKC-beta protein was reduced by 63+/-3.7% within 48 h. Co-transfection of an expression vector for MITF-M, the MITF isoform specific for pigment cells, or empty control vector with a full-length PKC-beta promoter-CAT (chloramphenicol acetyltransferase) reporter construct (PKC-beta/CAT) into Cos-7 cells showed >60-fold increase in CAT activity. Melanocytes abundantly also expressed MITF-A, as well as the MITF-B and MITF-H isoforms. However, in contrast with MITF-M, MITF-A failed to transactivate co-expressed PKC-beta/CAT or CAT constructs under the control of a full-length tyrosinase promoter. Together, these results demonstrate that MITF, specifically MITF-M, is a key transcription factor for PKC-beta, linking the PKC- and cAMP-dependent pathways in regulation of melanogenesis.

1-Methyl-3-isobutylxanthine↗

PubMed2005

Chronic wound fluid suppresses proliferation of dermal fibroblasts through a Ras-mediated signaling pathway.

Wound fluid collected from chronic venous leg ulcers (chronic wound fluid (CWF)) has been shown to inhibit the growth of dermal fibroblasts by interfering with cell-cycle progression from G1 into S phase. Specifically, CWF was shown to downregulate the levels of hyperphosphorylated retinoblastoma tumor-suppressor gene (Rb) and cyclin D1, known to be critical for entering the S phase of the cell cycle. To further elucidate the effects of CWF, a Ras-mediated signaling pathway involving the mitogen-activated protein kinase kinase (MEK), known to modulate the expression of these cell-cycle-regulatory proteins, was examined. Transient transfection of dermal fibroblasts with constitutively active Ras abrogated the growth suppressive effects of CWF on hyperphosphorylated Rb (ppRb) and cyclin D1. In contrast, an MEK inhibitor PD 98059 mimicked the effects of CWF on these cell-cycle-regulatory proteins. Concurrent treatment with PD 98059 and CWF produced additive effects. Taken together, these results suggest that CWF inhibits the growth of dermal fibroblasts at least in part by decreasing the level of active Ras, resulting in decreased levels of ppRb and cyclin D1. Therefore, a Ras-dependent signaling pathway may mediate the growth inhibitory effect of CWF, and reconstitution of Ras activity may overcome this growth inhibitory effect.

Apoptosis↗

PubMed2005

Modulation of vascular endothelial growth factor receptors in melanocytes.

Vascular endothelial growth factor (VEGF) is constitutively produced by keratinocytes, but has no known epidermal target cell. We now report that normal human melanocytes (Mc) maintained in serum-free, hormone-, and growth factor-supplemented medium lacking phorbol ester and choleragen constitutively express VEGF receptor-1 (VEGFR-1), VEGFR-2, and neuropilin-1. Furthermore, stimulation of Mc with VEGF165 isoform leads to phosphorylation of VEGFR-2, the receptor responsible for most of the VEGF-mediated effects in endothelial cells, suggesting that the receptor is functional. Interestingly, in Mc, VEGFR-2 expression is induced by ultraviolet irradiation and is downregulated by VEGF and tumor necrosis factor-alpha. Prolonged culture (>8 weeks) in the presence of phorbol ester abrogates VEGFR-2 expression, explaining previous reports that Mc do not express VEGFR-1 and VEGFR-2. These data suggest that VEGF may play a role in Mc behavior in skin.

Blotting, Western↗

PubMed2004

The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-beta on melanosomes.

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, alpha, beta, delta, epsilon and zeta isoforms of PKC are expressed, but uniquely PKC-beta activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-beta phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-beta, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-beta, revealed abundant PKC-beta, whereas PKC-alpha was not detected when immunoblot analysis was performed using antibody against PKC-alpha. Activation of PKC in melanocytes increased the level of PKC-beta co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-beta in the presence of UV-activated 1, 1'-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-beta and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-beta on the melanosome membrane, allowing PKC-beta to phosphorylate tyrosinase.

Base Sequence↗

PubMed2004

Topical application of a protein kinase C inhibitor reduces skin and hair pigmentation.

To determine whether inhibition of PKC-beta activity decreases pigmentation, paired cultures of primary human melanocytes were first pretreated with bisindolylmaleimide (Bis), a selective PKC inhibitor, or vehicle alone for 30 min, and then treated with TPA for an additional 90 min to activate PKC in the presence of Bis. Bis blocked the expected induction of tyrosinase activity by activation of PKC. Addition of a peptide corresponding to amino acids 501-511 of tyrosinase containing its PKC-beta phosphorylation site, a presumptive PKC-beta pseudosubstrate, gave similar results. To determine whether Bis reduces pigmentation in vivo, the backs of four shaved and depilated pigmented guinea pigs were UV irradiated with a solar simulator for 2 wk excluding weekends. Compared to vehicle alone, Bis (300 microM), applied twice daily to paired sites for various periods encompassing the irradiation period, decreased tanning. Bis also, although less strikingly, reduced basal epidermal melanin when topically applied twice daily, 5 d per wk, for 3 wk to shaved and depilated unirradiated skin. Moreover, topical application of Bis (100 microM) once daily for 9 d to the freshly depilated backs of 8-wk-old mice markedly lightened the color of regrowing hair. These results demonstrate that inhibiting PKC activity in vivo selectively blocks tanning and reduces basal pigmentation in the epidermis and in anagen hair shafts.

Animals↗

PubMed2003

Protein kinase C-beta-mediated complex formation between tyrosinase and TRP-1.

Tyrosinase, the key enzyme in melanogenesis, is activated when protein kinase C-beta (PKC-beta) phosphorylates the serine residues at amino acid positions 505 and 509. To further elucidate the mechanism by which phosphorylation of tyrosinase by PKC-beta leads to the activation of tyrosinase, a possible complex formation between phosphorylated tyrosinase and tyrosinase related protein-1 (TRP-1), a melanogenic protein suggested to influence tyrosinase activity, was investigated. Non-denaturing gel electrophoresis of melanocyte lysate revealed two molecular weight forms of TRP-1 and a monoclonal antibody against TRP-1 co-immunoprecipitated tyrosinase and TRP-1, suggesting that TRP-1 may be complexed with tyrosinase. Activation of PKC by treating melanocytes with phorbol 12,13-dibutyrate (PDBu) increased the level of tyrosinase co-immunoprecipitated with TRP-1; whereas a selective PKC inhibitor bisindolylmaleimide inhibited PDBu-induced increase in the level of tyrosinase co-immunoprecipitated with TRP-1. These results suggest that phosphorylation of tyrosinase by PKC-beta induces a complex formation between tyrosinase and TRP-1.

Cells, Cultured↗

PubMed2002

Purification, characterization, and cDNA cloning of rice class III chitinase.

Several chitinases were expressed in a rice cell suspension culture and detected in the medium. One of them, designated as RCB4, was isolated 248 fold from the culture filtrate to homogeneity by 70% ammonium sulfate precipitation, DEAE-cellulose, CM-cellulose, Sephadex G-75 column chromatography, and native gel slicing. RCB4 had a molecular mass of 32 kDa by SDS-PAGE. The optimum temperature was 40 degrees C, and 96% of its activity still remained at 60 degrees C. The optimum pH was 4, and 95% of its activity was maintained at pH 2. Using a substrate (GlcNAc)6, the Km and Vmax values of RCB4 were 0.53 mM and 11.1 mM/min, respectively. The N-terminal and internal amino acid sequences of RCB4 were determined to be VNSNLFRDYIGA and MALWA, respectively. A cDNA (C12523) clone that contained the N-terminal and internal amino acid sequences of RCB4 was obtained, sequenced, and renamed RCB41. RCB41 encoded 307 amino acid protein with a signal peptide of 25 amino acids and showed a 45% similarity to gladiolus chitinase GBC-a, one of the class III chitinase family. The expression of RCB4l in E. coli showed that RCB41 encodes a chitinase.

Amino Acid Sequence↗

PubMed2002

More on MITF.

Explore the source record for details and available documents.

Animals↗