Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

Rapid and reversible regulation of collagen XII expression by changes in tensile stress

We studied the expression of the fibril-associated collagen XII by fibroblasts cultured on attached (stretched) or floating (relaxed) collagen I gels. Accumulation of collagen XII in the medium as determined by semiquantitative immunoblotting was 8-16 times higher under stretched compared to relaxed conditions. Northern blot experiments showed that tensile stress controls collagen XII expression at the mRNA level. Tenascin-C mRNA levels were also influenced, whereas relative amounts of fibronectin and matrix metalloproteinase-2 mRNA were barely affected. The response to a change in tensile stress is rapid, since de novo biosynthesis of collagen XII was fully down-regulated 12 h after relaxation of a stretched culture. To demonstrate that the effect is also reversible, we mounted collagen gels with attached cells to movable polyethylene plugs. The cultures were relaxed or stretched at intervals of 24 and 48 h, and media samples were analyzed every 24 h. By ELISA, the amount of collagen XII secreted into the medium was found to increase or decrease in accordance with the tensile stress applied. This is evidence that the mechanical stimulus per se, rather than an indirect secondary effect, was responsible for the observed changes in collagen XII production.     

Linkage mapping and partial sequencing of 10 cDNA loci in the chicken

Ten cDNA clones derived from chicken spleen cell mRNA have been partially sequenced and the genes which encode the mRNAs have been located within the linkage map of the chicken genome. The sequences of five of these clones show strong homology to known mammalian genes, the remainder show little homology to sequences present in the current databases. Interestingly, one of these clones appears to be the chicken homologue of the mammalian peptide transporter gene TAP2 and is located within the major histocompatibility complex. Two other clones are homologous to genes involved in protein synthesis and these are tightly linked in chickens, as in mice. These results suggest that partial sequencing and mapping of clones from selective cDNA libraries may be an efficient way of adding candidate genes to the chicken linkage map and that on a local scale there may be some conservation of grouping of genes between chickens and mammalian species.     

Analysis of expressed sequence tags (EST) obtained from common carp,Cyprinus carpio L., head kidney cells after stimulation by two mitogens,lipopolysaccharide and concanavalin-A

A representative cDNA library from mRNA obtained from lipopolysaccharide and concanavalin-A-induced head kidney cells of carp, Cyprinus carpio, was constructed. Two hundred single pass and partially sequenced clones (AU183343 to AU183542) were generated from expressed sequence tags (ESTs) and these were searched for homology in the DDBJ/GENBANK with blastN and blastX programs. Clones matching known genes were classified according to their function and distribution. One hundred and twenty-nine genes showed homology with known genes in databases, whereas 71 (35.5%) clones did not show any significant homology to sequences in the public database. Known genes also showed homology to fish genes deposited in the database. Twenty-two clones (11%), encoding 16 different sequences, were identified as putative biodefense and oncogenes, associated with an immune response. High expression of lysozyme (3%) was detected. Putatively identified biodefense-related sequences such as Lectin type 2, MHC class II invariant chain, mcl-1a and lysozyme were aligned with known homologues from the database and the percentage identity determined. A time course evaluation of gene expression due to mitogen stimulation by RT-PCR revealed the above mentioned gene homologues were switched on early during the cell proliferation.     

Curcumin-induced differentiation of mouse embryonal carcinoma PCC4 cells.

Curcumin, a natural component of turmeric extracted from the rhizomes of Curcuma longa, is known to exhibit a number of biological properties. In the present study, curcumin, at low concentration, was shown to induce differentiation in embryonal carcinoma cell line PCC4. In response to curcumin, PCC4 cells ceased to proliferate and showed cell cycle arrest at G1 phase after 4 hours of treatment, followed by their differentiation which is characterized by increase of nuclear/cytoplasmic ratio. The expression of hsp 70 was also seen upon 8 h of curcumin treatment, and it remained constant up to 48 h. Differentiated cells also expressed a series of differentiation markers such as lamin A, well-established actin, and keratin cytoskeleton. We used mRNA differential display analysis to identify the genes that are regulated during curcumin-induced differentiation of PCC4 cells. We cloned and sequenced three partial cDNAs that were differentially expressed in normal and differentiated cells. Sequence comparison of one downregulated cDNA (Al) has shown homology to a gene present on mouse chromosome five, while the two upregulated cDNA (C1 and C7) are homologous to several mouse ESTs clones from organs of mesodermal origin. We have identified the full-length coding sequence of the Cl fragment with a putative amino acid sequence. Tissue-specific Northern with RNA from adult mouse organs with the C1 fragment alone showed hybridization with mRNA from several tissues, whereas the same Northern with only the coding sequence showed expression of C1 gene mainly in the adult kidney. Homology search revealed that C1 sequence is part of the 3' UTR and may be common to several genes expressed in many tissues. Thus, curcumin appears to differentiate embryonal carcinoma cell PCC4, and one of the upregulated genes seems to be expressed mainly in the adult kidney.     

Metabolic engineering of cultured tobacco cells

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5'-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.     

Differential gene expression during Trypanosoma cruzi metacyclogenesis

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.     

Identification of differentially expressed genes involved in the regression and development of the chicken Müllerian duct

Müllerian ducts of male chickens undergo regression around day 12 of incubation, but the underlining mechanisms remain unclear. The purpose of this study was to identify factors that contribute to regression of the Müllerian duct in the chicken. We first employed annealing control primer-based RT-PCR to screen candidate genes differentially expressed in the Müllerian ducts between male and female. Four differentially expressed genes (MSX2, GAL10, VCP and PLCH1) were partially sequenced. The expression of mRNA of the latter genes and MSX1 in the male and female Müllerian ducts were compared at 7.5, 8 and 9 days of incubation using semi-quantitative RT-PCR. The results indicated that both MSX1 and MSX2 mRNA was highly expressed in the male Müllerian duct at day 9 of incubation, whereas, PLCH1 mRNA was lower in the male duct at day 9 of incubation compared to that of the female duct. Although VCP mRNA was expressed in both left and right female Müllerian ducts, no expression was detected in the male duct. Whole mount in situ hybridyzation analysis showed that the expression of MSX1 and MSX2 mRNA were localized specifically in the mesenchymal cells of the male Müllerian duct at day 9 of incubation. In contrast, VCP mRNA expression was observed in both mesenchymal and epithelial cells of the female Müllerian duct but not detected in the male duct. These results suggest that both up-regulation of MSX1 and MSX2 mRNA expression is involved in the regression of the Müllerian duct in male chicken embryo, whereas VCP expression is involved in development of the female duct.