Developmental Regulation of Focal Contact Protein Expression in Human Melanocytes

Focal contacts are transmembrane links between the extracellular matrix and the actin cytoskeleton that play a critical role in directed cell migration, adhesion, and normal growth. Several different component proteins of the focal contact show develop-mentally dependent changes in expression, suggesting that this is an important mechanism by which focal contact formation is controlled during embryogenesis. In this report we examine the expression of focal contact-associated proteins in human fetal and neonatal melanocytes using Western blotting. We show that expression of paxillin, a 69-kDa vinculin binding protein, is fourfold higher in neonatal melanocytes than in fetal melanocytes. Further, we show that talin, a high molecular weight structural protein that links integrins to the actin cytoskeleton, is proteolytically cleaved in fetal, but not in neonatal melanocytes. Immunofluorescence microscopy of cells grown on fibronectin confirmed the presence of paxillin, talin, and vinculin at the ends of actin stress fibers at presumptive focal contacts in melanocytes. Adhesion experiments to extracellular matrix ligands revealed significant differences in adhesion of fetal and neonatal melanocytes to fibronectin. The developmentally specific changes in focal contact protein expression observed suggest that this may be an important mechanism by which focal contact assembly is controlled in human melanocytes during development.     

HSV-1感染对神经胶质瘤细胞神经生长因子及其受体表达的影响

神经生长因子(NGF)主要由神经胶质细胞产生,通过特异的靶细胞表面的神经生长因子受体介导产生生物学效应,与神经细胞的生长发育、分化和凋亡等密切相关。单纯疱疹病毒1型(HSV-1)作为一种嗜神经病毒,易造成神经细胞、神经胶质细胞凋亡或死亡。本实验以U251人神经胶质瘤细胞为研究对象,观察HSV-1感染致U251细胞凋亡的过程中NGF及其受体的变化情况。结果发现U251细胞是HSV-1的容许细胞;HSV-1感染致U251细胞凋亡过程中,NGF及其低亲和力受体p75NTR出现表达强度随时间先增强后减弱的趋势,而高亲和受体Tr-kA持续低表达。推测HSV-1感染致神经细胞凋亡中可能调控了神经营养因子的表达。     

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.     

神经元限制性沉默因子在NTera2/CloneD1细胞向神经元分化模型中表达的检测

目的:建立NTera2/CloneD1细胞向神经元分化的模型,检测神经元限制性沉默因子(NRSF)经分化培养基诱导后表达的变化。方法:收集正常培养的NTera2/CloneD1细胞及经全反式维甲酸(RA)、阿糖胞苷(AraC)、尿苷分阶段诱导共28 d的细胞,显微镜下观察诱导前后细胞的形态学变化;免疫荧光法检测NTera2/CloneD1细胞诱导前后干性标志Nestin、Sox2和成熟神经元特异性标志NF-200、β-tubulinⅢ的表达情况;应用RT-PCR和免疫荧光法对NRSF进行mRNA和蛋白水平的检测。结果:显微镜下观察到正常培养的NTera2/CloneD1细胞呈克隆样生长,经分化培养基诱导后的NTera2/CloneD1细胞表现出典型的神经元样细胞形态。免疫荧光检测表明,未诱导的NTera2/CloneD1细胞表达神经干细胞的标志Sox2、Nestin,不表达成熟神经元特异性蛋白NF-200、β-tubulinⅢ;而经RA等诱导分化的细胞则不表达Sox2、Nestin,表达NF-200、β-tubulinⅢ。RT-PCR和免疫荧光检测显示,NRSF在诱导分化后的NTera2/CloneD1细胞中的表达量显著降低。结论:建立了NTera2/CloneD1细胞向神经元分化的模型,NRSF在诱导后的NTera2/CloneD1细胞中表达量显著下调,提示NTera2/CloneD1细胞在诱导过程中可能通过下调NRSF,使受到NRSF负性调控的神经元特异性蛋白启动表达并上调,进而实现NTera2/CloneD1细胞向神经元的定向分化。     

Regulation of the Cell Cycle at the G2/M Boundary in Metastatic Melanoma Cells by 12-O-Tetradecanoyl Phorbol-13-acetate (TPA) by Blocking p34Kinase Activity

12-O-Tetradecanoyl phorbol-13-acetate (TPA) inhibits the growth of most malignant melanoma cells but stimulates the growth of normal human melanocytes. We previously showed that addition of TPA inhibits the growth of the human metastatic melanoma cell line, Demel, by blocking cells at both the G1/S and G2/M cell cycle transitions (D. L. Coppocket al.,1992,Cell Growth Differ.3, 485–494). To examine the G2/M transition, we developed a method to synchronize the cells in early S phase using Lovastatin and mevalonate, followed by treatment with hydroxyurea (HU). TPA (30 nM) was effective in blocking cells from entering mitosis and reentering G1 when added up to the end of G2. These cells arrested in G2. Examination of the levels of cyclins A and B1 demonstrated that the levels of these cyclins were not limiting for entrance into M. However, the addition of TPA blocked the increase in p34^cdc2/cyclin B1 kinase activity. In cells treated with TPA, most p34^cdc2was found in the slowly migrating forms on Western blots, which contained increased levels of phosphotyrosine. In addition, the level of the cyclin-dependent kinase inhibitor p21^Cip1/Waf1, but not of p27^Kip1, was increased. We examined the expression of protein kinase C (PKC) isoforms in Demel cells using Western blots to understand which types were involved in the G2 arrest. Demel cells expressed the PKC α, βI, βII, δ, ε, ι/λ, ζ, and μ isozymes. PKC η and PKC θ were not detected. Addition of TPA did not completely down regulate any PKC isozymes over a 12-h period in these synchronized cells. PKC α, βI, βII, δ, and ε isozymes were translocated to the membrane fraction from the cytosolic fraction when treated with TPA. PKC δ appeared as a doublet and the addition of TPA shifted a majority to the slower migrating form. The level of PKC μ was constant; however, a slow mobility form was observed in TPA-treated cells. This reduced mobility was at least partially due to phosphorylation. Thus, the arrest of growth in G2 appears to be due to the inhibition of the p34^cdc2kinase activity which is associated with the increased expression of p21^Cip1/Waf1and increased phosphorylation on tyrosine of p34^cdc2. This arrest, in turn, is associated with a shift of PKC isozymes PKC α, PKC βI, PKC βII, PKC δ, PKC ε, and PKC μ to the membrane fraction which is induced by addition of TPA.     

In Situ and In Vitro Expression of Protein Kinase C Alpha in Human Melanocytes

Protein kinase C (PKC) is a multigene family of at least 12 isoforms involved in the transduction of extracellular signals. We investigated whether PKC-α, a major isoform known to be relatively abundant in brain tissue, is increased in human melanocytes relative to keratinocytes in vitro and in situ. Immunohistochemical staining for PKC-α in frozen neonatal human foreskin exhibited intermittent 2–3+ staining along the basal cell layer consistent with melanocytes, and 0–1+ staining of keratinocytes (on a scale of 0–3). Microscopic densitometry of the intermittent cellular staining was at least 3-fold greater than that of adjacent keratinocyte cell cytoplasm. Sequential frozen sections revealed similar intermittent cell staining with PKC-α and Mel-5 (tyrosinase related protein-1), known to specifically react with melanocytes. Northern blot analysis with a specific cDNA probe for PKC-α showed strong PKC-α mRNA expression in cultured melanocytes, whereas PKC-α mRNA in cultured non-stratifying keratinocytes was expressed at low levels. Western blot analysis revealed a prominent PKC-α band at approximately 80 kDa in melanocytes as opposed to a weak band in keratinocytes. Densitometry of the northern and western blots revealed that melanocytes had at least 10-fold more PKC-α mRNA and approximately 6-fold more PKC-α protein expression than keratinocytes. Total PKC activity measured in vitro revealed that melanocytes had 5-fold more activity than keratinocytes. The marked difference in melanocyte and keratinocyte expression of PKC-α provides further evidence for cell type specificity in the balance of PKC-α expression and may implicate differential PKC isoform signaling pathways in neuro-ectodermally derived cells.     

The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low-serum medium deprived of phorbol esters, cholera toxin, and other non-physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue-type plasminogen activator and utilised it to generate cell-bound plasmin. No urokinase-type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines. The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.     

hnRNP A2/B1在人永生化表皮HaCaT细胞凋亡过程中的表达及其调控作用研究

该文以姜黄素诱导人永生化表皮HaCaT细胞凋亡为基础,对hnRNP A2/B1在核基质中的存在、分布及其与细胞凋亡相关基因产物的共定位及相互作用关系进行了研究。蛋白质印迹结果显示,hnRNP A2/B1存在于HaCaT细胞核基质蛋白组分中,在经过姜黄素处理后,表达下调;激光共聚焦显微镜观察显示,hnRNP A2/B1在HaCaT细胞中分别与Fas、p53和Bax等基因产物具有共定位关系,姜黄素处理后其共定位区域出现由核膜或核仁向胞质转移的趋势。GST pull-down实验证实,hnRNPA2/B1分别与Fas、p53和Bax有直接相互作用关系。结果表明,hnRNPA2/B1作为一种核基质蛋白,通过与细胞凋亡相关基因产物的相互作用参与HaCaT细胞的凋亡诱导调控过程,这对深入认识核基质蛋白在细胞凋亡过程中的调控机制具有重要意义。     

Luminol-Enhanced Chemiluminescent Response of Human Melanocytes and Melanoma Cells to Hydrogen Peroxide Stress

The response of human melanocytes and melanoma cells to hydrogen peroxide stress was measured. Cells were exposed to glucose/glucose oxidase or free H₂O₂ and reactive oxygen species measured by luminol-enhanced chemiluminescence. The response was distinctly different between the two types and the addition of superoxide dismutase to melanoma cells paradoxically enhanced the chemiluminescent signal. These findings coupled with other known differences between the way these two types of cells handle oxidative stress at a molecular level suggests that a therapeutic window may be avail-able for exploitation.     

Capsaicin-enhanced Ribosomal Protein P2 Expression in Human Intestinal Caco-2 Cells

On the basis of transepithelial electrical resistance (TER) measurements, we found that capsaicin (100 μM)-treated human intestinal Caco-2 cells show a momentary increase in tight-junction (TJ) permeability (decrease in TER) followed by a complete recovery. We used proteome analysis to search for proteins that are associated with the recovery of TJ permeability in capsaicin-treated Caco-2 cells. A protein with a relative molecular mass of 14 kDa was found to be expressed more highly in capsaicin-treated cells than in nontreated cells. Mass spectrometry and sequence analyses revealed that the protein that is expressed significantly upon capsaicin treatment is the ribosomal protein P2; its cDNA sequence was identical to that found in the human genome database. An increase in the amount of cellular filamentous actin (F-actin) was shown after 8 h of incubation with capsaicin. It has been reported that P2 activates elongation factor 2, which stabilizes F-actin filaments, and that the depolymerization of F-actin is associated with the increase in TJ permeability (decrease in TER). Consequently, these results suggest that P2 plays an important role in the recovery of the TJ permeability in capsaicin-treated human intestinal cells. An erratum to this article is available at .     

Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation

Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1^−/− chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

核转录因子Foxp3在小鼠黑色素瘤细胞中的表达

目的:检测小鼠黑色素瘤细胞中核转录因子Foxp3的表达。方法:用TRIzol试剂提取小鼠黑色素瘤细胞RNA,通过实时荧光定量PCR检测小鼠黑色素瘤细胞中Foxp3 mRNA的表达,通过Western印迹和流式细胞术检测小鼠黑色素瘤细胞中Foxp3蛋白的表达,通过免疫荧光检测小鼠黑色素瘤细胞中Foxp3分子的表达。结果:在小鼠黑色素瘤细胞中存在Foxp3的mRNA转录和分子表达,免疫荧光显示Foxp3定位于黑色素瘤细胞的胞核及核周部位。结论:证实了小鼠黑色素瘤细胞表达Foxp3,将为临床黑色素瘤的免疫治疗提供新的靶标和治疗策略。     

Regulation of ERK1/2 activity upon contact inhibition in fibroblasts

Contact inhibition is a crucial mechanism regulating proliferation in vitro and in vivo. Despite its generally accepted importance for maintaining tissue homeostasis knowledge about the underlying molecular mechanisms of contact inhibition is still scarce. Since the MAPK ERK1/2 plays a pivotal role in the control of proliferation, we investigated regulation of ERK1/2 phosphorylation which is downregulated in confluent NIH3T3 cultures. We found a decrease in upstream signaling including phosphorylation of the growth factor receptor adaptor protein ShcA and the MAPK kinase MEK1/2 in confluent compared to exponentially growing cultures whereas involvement of ERK1/2 phosphatases in ERK1/2 inactivation is unlikely. Treatment of confluent, serum-deprived cultures with PDGF-B resulted in similar phosphorylation of ERK1/2 and induction of DNA-synthesis as detected in sparse, serum-deprived cultures. In contrast, ERK1/2 phosphorylation and DNA-synthesis could not be stimulated in confluent, serum-deprived cultures exposed to EGF. Our data indicate that PDGFR- and EGFR signaling are differentially inhibited in confluent cultures of NIH3T3 cells.     

Over‐expression of MSG1 Transcriptional Co‐activator Increases Melanin in B16 Melanoma Cells: A Possible Role for MSG1 in Melanogenesis

MSG1 is a 27 kDa nuclear protein that is expressed strongly in melanotic B16 melanoma cells but very weakly in amelanotic B16 cells. Transient expression of B16 cells with an expression vector for MSG1 resulted in an increase in levels of the enzyme dopachrome tautomerase but not tyrosinase, as detected by western blotting. Stable transfection of B16 melanoma cells with plasmids containing the full length MSG1 or its deletion mutants, however, generated cell lines that showed an increase in levels of tyrosinase, dopachrome tautomerase and cellular melanin when compared with control transfected cells. Our results suggest that MSG1 plays an important role in melanogenesis, by regulating the levels of the enzymes of the pigmentary system via tyrosinase and dopachrome tautomerase.     

膜蛋白KCNN1在昆虫细胞Sf9中的表达与纯化

目的:利用昆虫细胞表达系统真核表达并纯化小电导钙激活钾离子通道蛋白1(KCNN1)。方法:以基因重组方法构建杆状病毒穿梭质粒reBacmid-KCNN1,将其转染至杆状病毒/Sf9细胞表达系统表达目的蛋白,并用Western印迹鉴定KCNN1的表达水平;用Ni-IDA-Sepharose CL-6B亲和层析柱纯化裂解细胞上清中的KCNN1,并用Western印迹鉴定纯化结果。结果:KCNN1在Sf9细胞中高效表达,通过亲和层析获得了纯化的KCNN1。结论:膜蛋白KCCN1在昆虫细胞Sf9中的表达与纯化,为深入研究其分子生物学功能提供了材料,也为全长膜蛋白的体外表达提供了一套可借鉴的实验方法。     

Epidermal Growth Factor Inhibits Gap Junctional Communication and Stimulates Serine-Phosphorylation of Connexin43 in WB Cells by a Protein Kinase C-Independent Mechanism

Epidermal growth factor (EGF) stimulated the phosphorylation of connexin43 (Cx43) in WB cells as evidenced by the formation of multiple irnmunoreactive Cx43 proteins of higher molecular mass which were abolished by treatment with alkaline phosphatase. Phosphorylation of Cx43 occurred within 10 min of EGF stimulation, was sustained for 1 h, and was associated with almost complete inhibition of gap junctional communication in these cells. EGF-induced phosphorylation and communication inhibition were retained in cells pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete protein kinase C. These results show that the EGF inhibition of communication is tightly linked to protein kinase C-independent phosphorylation of Cx43. Further, Cx43 phosphorylated in the presence of EGF did not react with phosphotyrosine antibodies and in ³²P_i incorporation experiments was shown to contain only phosphoserine indicating that the tyrosine kinase activity of the EGF receptor was not directly involved.