Genomic organization, sequence analysis and transcriptional regulation of the human MCP-4 chemokine gene (SCYA13) in dermal fibroblasts: a comparison to other eosinophilic beta-chemokines.

The eosinophil chemotactic beta-chemokine MCP-4 is assumed to be involved in the accumulation of eosinophils characteristic for eosinophilic inflammatory diseases. We here describe the genomic organisation (3 exons of 138, 115 and 578 bp, 2 introns of 867 and 437 bp and 1.4 kb of regulatory sequences from the immediate 5' upstream region), sequence (genomic and transcribed) and mRNA expression of the human MCP-4 gene in dermal fibroblasts. Among the promoter elements potentially regulating MCP-4 gene expression and/or mediating the effects of anti-inflammatory drugs we identified consensus sequences known to interact with nuclear factors like NF-IL6, AP-2, a NF-kappaB like consensus sequence, gamma-interferon- response and YY-1 elements as well as glucocorticoid response elements. Like MCP-3, MCP-4 mRNA expression in dermal fibroblasts is upregulated by TNF-alpha, IL-1alpha, IFN-gamma or IL-4 and differs from RANTES and eotaxin mRNA expression in its response to IFN-gamma and/or IL-4.

The CXC receptor 2 is overexpressed in psoriatic epidermis.

The CXC chemokines interleukin-8 and GRO/melanoma growth-stimulatory activity (GRO-alpha) are potent activators of neutrophils and lymphocytes, but also stimulate growth and differentiation of nonhematopoietic cells like keratinocytes, fibroblasts, and melanocytes. High mRNA and protein levels have been detected in psoriatic epidermis. Chemokine activation of target cells is mediated by specific receptors and two CXC receptors have been described with similar affinity for interleukin-8 but different affinities for GRO-alpha. In this study, we examined the expression of both CXCR1 and CXCR2 in psoriatic tissue, identifying the target cells of chemokine activation in psoriasis. By immunohistochemistry and in situ hybridization, as confirmed by northern blot analysis and reverse transcriptase polymerase chain reaction, we could detect expression of the CXCR2 in suprabasal lesional psoriatic keratinocytes but not in healthy skin. The CXCR1 could not be localized in psoriatic keratinocytes with immunohistochemistry and in situ hybridization, but infiltrating cells in the dermal compartment expressed both types of receptors. These data suggest that in addition to neutrophil activation by both CXCR1 and CXCR2, activation of keratinocytes mediated by CXCR2 could contribute to the characteristic epidermal changes observed in psoriasis.

Genomic organization, sequence, and transcriptional regulation of the human eotaxin gene.

Eotaxin is an eosinophil specific beta-chemokine assumed to be involved in eosinophilic inflammatory diseases such as atopic dermatitis, allergic rhinitis, asthma and parasitic infections. Its expression is stimulus- and cell-specific. We here describe the genomic organisation (3 exons of 132, 112 and 542 bp and 2 introns of 1211 and 378 bp) and sequence including 3 kb of DNA from the immediate 5' upstream region of the human eotaxin gene. Among the regulatory promoter elements potentially regulating eotaxin gene expression and/or mediating the effects of anti-inflammatory drugs we identified consensus sequences known to interact with nuclear factors like NF-IL6, AP-1, a NF-kappa-B like consensus sequence and gamma-interferon- as well as glucocorticoid response elements.

Increased eotaxin-mRNA expression in non-atopic and atopic nasal polyps: comparison to RANTES and MCP-3 expression.

Eosinophilic tissue infiltration of nasal mucosa typical for allergic rhinitis and chronic polypous sinusitis may be due to chemotactic activity of chemokines specific for eosinophils. The CC-chemokines eotaxin, RANTES and MCP-3 have been postulated to be involved in the recruitment of eosinophils to certain inflamed tissues. To explore their possible role in chronic polypous sinusitis we examined eotaxin-, RANTES- and MCP-3-gene expression in human nasal polyps and normal human nasal mucosa of patients undergoing endonasal surgery for treatment of chronic polypous sinusitis. Using gene-specific primers in semi-quantitative reverse-transcriptase polymerase-chain-reaction experiments we found elevated expression of eotaxin- and RANTES-mRNA but no MCP-3-mRNA in non-atopic and atopic nasal polyps when compared to normal nasal mucosa.

Fibroblasts but not epithelial cells obtained from human nasal mucosa produce the chemokine RANTES.

RANTES is a chemokine that was already found in tissues obtained from nasal polyps of patients suffering from chronic polypous sinusitis. Its cellular origin is as yet unknown. The aim of this study was to investigate whether human nasal mucosa fibroblasts and epithelial cells are capable to produce RANTES. Fibroblasts and epithelial cells, obtained from healthy human nasal mucosa, were cultured. Expression of RANTES-mRNA and secretion of RANTES-protein in supernatants was investigated after stimulation with 50 ng/ml Tumour Necrosis Factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interferon-g (IFN-gamma), lipopolysaccharide (LPS), phorbolymyristate acetate (PMA) and serum-free medium (SFM) for 24 h. Cultivated nasal fibroblasts either expressed RANTES-mRNA or secreted RANTES protein upon TNF-alpha, IL-1 beta and IFN-gamma stimulation. The amounts of RANTES-protein production ranged from 23 ng/ml (PMA) to 198 ng/ml (TNF-alpha). Nasal epithelial cells expressed RANTES-mRNA only after stimulation with PMA. Secretion of significant amounts of RANTES protein were not detected in the supernatants from nasal epithelial cells. We conclude that nasal fibroblasts but not epithelial cells could be a cellular source of RANTES in nasal mucosa or in secretions of patients suffering from diseases, where eosinophilic tissue infiltration represents a characteristic histopathological feature.

Human dermal fibroblasts express eotaxin: molecular cloning, mRNA expression, and identification of eotaxin sequence variants.

Recently we discovered and purified a novel beta-chemokine with eosinophil specific chemotactic activity from supernatants to long-term TNF-alpha stimulated dermal fibroblasts. Using degenerated specific oligonucleotides based on partial amino acid sequence data and a PCR protocol, we obtained different clones sharing high sequence homology with this novel chemokine and with human eotaxin cDNA. Semi-quantitative RT-PCR experiments using eotaxin-specific primers indicate low constitutive eotaxin mRNA expression in human dermal fibroblasts which is upregulated by IL-1 alpha and TNF-alpha within 6 hrs and modulated by IFN-gamma. While IL-1 alpha-induced eotaxin mRNA accumulation is transient, long-term stimulation with TNF-alpha resulted in a further increase of eotaxin mRNA.

Co-localized overexpression of GRO-alpha and IL-8 mRNA is restricted to the suprapapillary layers of psoriatic lesions.

Epidermal infiltration by polymorphonuclear cells is a prominent feature in psoriatic lesions. Expression of neutrophil-specific chemoattractants by lesional keratinocytes could play an important role in the regulation of this infiltration process. We therefore examined the mRNA expression of GRO-alpha, a well-characterized peptide with neutrophil-specific activation profile in psoriatic lesions by in situ hybridization. Clusters of clearly detectable and in some cases highly abundant GRO-alpha hybridization signals could be demonstrated in the differentiated layers of psoriatic epidermis. The signals were clearly associated with keratinocytes, with no nearby neutrophils detectable by microscopic examination. When additional tissue sections of GRO-alpha-expressing lesions were examined with an interleukin-8/neutrophil-activating peptide-8 (IL-8)-specific anti-sense probe, IL-8 expression was detectable and confined to areas also expressing GRO-alpha. Expression of both GRO-alpha and IL-8 is focally upregulated by an as yet unknown mechanism in lesional psoriatic keratinocytes, ultimately leading to neutrophil tissue infiltration. We suggest that the focal expression of GRO-alpha and IL-8 in the epidermal layers above the dermal papillae may be involved in the "squirting papilla" reaction described as a characteristic feature of psoriatic plaque-type lesions.

Detection of the chemokine RANTES in cytokine-stimulated human dermal fibroblasts.

A novel family of structurally and functionally related polypeptides has recently been detected that are now referred to as chemokines. Within this family, a peptide with the acronym RANTES was shown to be chemotactic for memory T cells, monocytes, and eosinophilic and basophilic granulocytes, thus suggesting it plays an important role in chronic inflammatory and allergic diseases. Murine monoclonal antibodies as well as cDNA probes specific for human RANTES were raised and extensively characterized. With these antibodies, stimulated human dermal fibroblasts were shown to express intracellular RANTES peptide by immunocytochemistry. Furthermore, similar kinetics could be demonstrated in fibroblasts for both RANTES mRNA expression and secretion of RANTES peptide using Northern blot hybridization and sandwich-enzyme-linked immunosorbent assay, respectively. RANTES expression was induced upon stimulation with tumor necrosis factor-alpha as well as with interleukin-1 alpha and -beta in a concentration- and time-dependent manner. These results reinforce the role of both resident and circulating cells in the production and release of RANTES and their participation in inflammatory processes.

A glutamine residue in the membrane-associating domain of the bovine papillomavirus type 1 E5 oncoprotein mediates its binding to a transmembrane component of the vacuolar H(+)-ATPase.

The 44-amino-acid E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. It is a highly hydrophobic polypeptide which dimerizes and localizes to the Golgi apparatus and endoplasmic reticulum membranes. Recent evidence suggests that E5 modulates the phosphorylation and internalization of the epidermal growth factor and colony-stimulating factor 1 receptors and constitutively activates platelet-derived growth factor receptors in C127 and FR3T3 cells. Although no direct interaction with these growth factor receptors has yet been identified, the E5 oncoprotein has been shown recently to interact with the hydrophobic 16-kDa component of the vacuolar H(+)-ATPase (16K protein) [D. J. Goldstein, M. E. Finbow, T. Andresson, P. McLean, K. Smith, V. Bubb, and R. Schlegel, Nature (London) 352:347-349, 1991]. In the current study, we have further analyzed the E5-16K protein complex by fast protein liquid chromatography and shown that each E5 dimer appears to bind two 16K proteins. In order to define the specific amino acid residues of E5 which participate in this binding, mutated E5 epitope fusion proteins were analyzed for their ability to coprecipitate 16K protein. Transformation-defective mutants containing amino acid substitutions within the short hydrophilic carboxyl-terminal domain retained the ability to associate with the 16K protein. However, E5 mutants lacking the glutamine residue in the hydrophobic domain were markedly inhibited in 16K protein binding. Most interestingly, the placement of a glutamine in several random hydrophobic sequences facilitated 16K protein binding, defining this residue as a potential binding site for the 16K protein component of the proton pump and exemplifying the critical role of hydrophilic amino acids for mediating specific interactions between transmembrane proteins.

The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine.

The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl-terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability.

Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16.

To examine the biological properties of the bovine papillomavirus type 1 (BPV) and human papillomavirus type 16 (HPV16) E5 genes, each was cloned separately into a retroviral expression vector and helper-free recombinant viruses were generated in packaging cell lines. The BPV E5 retroviruses efficiently caused morphologic and tumorigenic transformation of cultured lines of murine fibroblasts, whereas the HPV16 E5 viruses were inactive in these assays. In contrast, infection of the p117 established line of murine epidermal keratinocytes with either the BPV or the HPV16 E5 retrovirus resulted in the generation of tumorigenic cells. Pam212 murine keratinocytes were also transformed to tumorigenicity by the HPV16 E5 gene but not by the gene carrying a frameshift mutation. These results establish that the HPV16 E5 gene is a transforming gene in cells related to its normal host epithelial cells.

Biological properties of the deer papillomavirus E5 gene in mouse C127 cells: growth transformation, induction of DNA synthesis, and activation of the platelet-derived growth factor receptor.

We determined the biological activities of the 44-amino-acid deer papillomavirus (DPV) E5 protein in mouse C127 cells. The DPV E5 gene can induce focus formation, stable and acute morphologic transformation, and DNA synthesis, and it activates the platelet-derived growth factor (PDGF) beta receptor as assessed by increased constitutive tyrosine phosphorylation of mature and precursor receptor forms. Thus, the DPV E5 protein has biological activities similar to those of the closely related E5 protein from bovine papillomavirus type 1. Moreover, like the bovine papillomavirus type 1 E5 protein, the DPV E5 protein shares a short region of sequence similarity with the B chain of PDGF. These results show that activation of the PDGF receptor is a general property of fibropapillomavirus E5 proteins, lending support to our previous proposal (L. Petti, L. Nilson, and D. DiMaio, EMBO J. 10:845-855, 1991) that activation of the PDGF receptor may play a central role in transformation of fibroblasts by E5 proteins.

Duplication of enhancer sequences in human papillomavirus 6 from condylomas of the mamilla.

Human papillomavirus (HPV) 6 usually induces tumors of the genital, oral, or laryngeal mucosa. An HPV 6-related DNA of 8.2 kb was detected in an extrachromosomal state in atypically located condylomas of the mamilla and was molecularly cloned. The identity of the cloned HPV DNA and the viral DNA in the biopsy was confirmed by comparative Pstl cleavage analysis, which showed typical HPV 6 DNA fragment patterns except for 0.2-kb larger B fragments. Sequencing revealed an exact 236-bp duplication encompassing nucleotides 7681 to 7896 of HPV 6b. This tandem repeat is just upstream from the putative early promoter and contains a 20-bp insertion at position 7720, which constitutes an enhancer element described by R. F. Rando, W. D. Lancaster, P. Han, and C. Lopez (Virology 155, 545-556, 1986). A Hinfl-Pstl fragment containing the whole duplication was cloned into an enhancer-dependent CAT expression vector and led to three- to sevenfold increased CAT activity when compared with the monomeric sequence in C127 cells and BPV1 transformed C127 cells. This indicates that the duplication within the HPV 6 isolate from the mamilla may influence early gene expression and possibly tissue tropism.