scholarly journals DNA-mediated gene transfer into adult rat hepatocytes in primary culture.

1990 ◽  
Vol 10 (2) ◽  
pp. 689-695 ◽  
Author(s):  
R A Rippe ◽  
D A Brenner ◽  
H L Leffert
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Primary Culture ◽  
Tissue Specificity ◽  
Transforming Growth Factor ◽  
Rat Hepatocytes ◽  
Transforming Growth Factor Alpha ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Factor Alpha

Proliferation-competent and differentiation-competent adult rat hepatocytes in primary culture were investigated for their ability to express reporter genes (firefly luciferase, bacterial chloramphenicol acetyltransferase, and bacterial beta-galactosidase) driven by tumor virus or eucaryotic promoters that vary in transcriptional efficiency and tissue specificity. Supercoiled plasmid DNA molecules were introduced into the cells by the calcium phosphate coprecipitation protocol of C. Chen and H. Okayama (Mol. Cell. Biol. 7:2745-2752, 1987). Reporter gene expression was virtually restricted to hepatocytes and was efficient (2 to 20% of the cells). The patterns and absolute levels of reporter gene expression depended on assay conditions employed (plasmid concentration [optimal at 2.4 micrograms of DNA per ml] and duration of exposure [optimal between 5 and 10 h]), culture growth cycle stages (lag, log, or stationary phase), properties and tissue specificity of the promoter(s) tested, and composition (and timing of fluid change) of the culture medium with or without the hepatocyte mitogen human transforming growth factor-alpha. Initial observations suggest that during hepatocellular growth transitions, human transforming growth factor-alpha differentially regulates exogenously introduced promoters associated with hepatocyte-specific function and proliferation. These findings provide a simple, fast, and powerful approach to analyzing the molecular and cellular biology of hepatocyte growth control.

DNA-mediated gene transfer into adult rat hepatocytes in primary culture

1990 ◽  
Vol 10 (2) ◽  
pp. 689-695
Author(s):  
R A Rippe ◽  
D A Brenner ◽  
H L Leffert
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Primary Culture ◽  
Tissue Specificity ◽  
Transforming Growth Factor ◽  
Rat Hepatocytes ◽  
Transforming Growth Factor Alpha ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Factor Alpha

Proliferation-competent and differentiation-competent adult rat hepatocytes in primary culture were investigated for their ability to express reporter genes (firefly luciferase, bacterial chloramphenicol acetyltransferase, and bacterial beta-galactosidase) driven by tumor virus or eucaryotic promoters that vary in transcriptional efficiency and tissue specificity. Supercoiled plasmid DNA molecules were introduced into the cells by the calcium phosphate coprecipitation protocol of C. Chen and H. Okayama (Mol. Cell. Biol. 7:2745-2752, 1987). Reporter gene expression was virtually restricted to hepatocytes and was efficient (2 to 20% of the cells). The patterns and absolute levels of reporter gene expression depended on assay conditions employed (plasmid concentration [optimal at 2.4 micrograms of DNA per ml] and duration of exposure [optimal between 5 and 10 h]), culture growth cycle stages (lag, log, or stationary phase), properties and tissue specificity of the promoter(s) tested, and composition (and timing of fluid change) of the culture medium with or without the hepatocyte mitogen human transforming growth factor-alpha. Initial observations suggest that during hepatocellular growth transitions, human transforming growth factor-alpha differentially regulates exogenously introduced promoters associated with hepatocyte-specific function and proliferation. These findings provide a simple, fast, and powerful approach to analyzing the molecular and cellular biology of hepatocyte growth control.

scholarly journals IGF-I production by adult rat hepatocytes is stimulated by transforming growth factor-alpha and transforming growth factor-beta1

1999 ◽  
pp. 577-582 ◽  
Author(s):  
A Voci ◽  
M Arvigo ◽  
M Massajoli ◽  
S Garrone ◽  
C Bottazzi ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Rat Hepatocytes ◽  
Transforming Growth Factor Alpha ◽  
Cultured Hepatocytes ◽  
Transforming Growth Factor Beta1 ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Igf I ◽  
Factor Alpha

Previously, we have observed that epidermal growth factor (EGF), a potent mitogen for cultured hepatocytes, stimulates the production of IGF-I and IGF-binding proteins (IGFBPs) by cultured hepatocytes from adult rats. This study was undertaken to investigate the possibility that other growth factors of hepatic origin could specifically be involved in the regulation of IGF-I and IGFBP expression. The effects of transforming growth factor-alpha (TGF-alpha), through EGF receptors to induce a mitogenic response, and transforming growth factor-beta1 (TGF-beta1), produced by non-parenchymal liver cells and able to inhibit hepatocyte proliferation in vivo and in culture, have been studied in cultured adult rat hepatocytes. Our results demonstrate that TGF-alpha and TGF-beta1 significantly stimulate IGF-I and IGFBP secretion by cultured hepatocytes but no change in the abundance of IGF-I and IGFBP mRNAs was observed with respect to controls. Cycloheximide is able to inhibit both basal and TGF-stimulated release of IGF-I and a similar effect was elicited by octreotide, the somatostatin analog, known to directly affect hepatic IGF-I gene expression. Our findings show the role of the liver in the secretion of IGF-I and IGFBPs, not only under endocrine and nutritional control but also under autocrine and paracrine control.

MOLECULAR CONTROL OF ARTICULAR CARTILAGE DEGENERATION BY TRANSFORMING GROWTH FACTOR ALPHA

2008 ◽  
Vol 31 (4) ◽  
pp. 2
Author(s):  
Tom Appleton ◽  
Shirine Usmani ◽  
John Mort ◽  
Frank Beier
Keyword(s):  
Gene Expression ◽  
Articular Cartilage ◽  
Growth Factor ◽  
P38 Mapk ◽  
Transforming Growth Factor ◽  
Cartilage Degeneration ◽  
Signalling Pathways ◽  
Transforming Growth Factor Alpha ◽  
Factor Alpha ◽  
Articular Cartilage Degeneration

Background: Articular cartilage degeneration is a hallmark of osteoarthritis (OA). We previously identified increased expression of transforming growth factor alpha (TGF?) and chemokine (C-C motif) ligand 2 (CCL2) in articular cartilage from a rat modelof OA (1,2). We subsequently reported that TGF? signalling modified chondrocyte cytoskeletal organization, increased catabolic and decreased anabolic gene expression and suppressed Sox9. Due to other roles in chondrocytes, we hypothesized that the effects ofTGF? on chondrocytes are mediated by Rho/ROCK and MEK/ERK signaling pathways. Methods: Primary cultures of chondrocytes and articularosteochondral explants were treated with pharmacological inhibitors of MEK1/2(U0126), ROCK (Y27632), Rho (C3), p38 MAPK (SB202190) and PI3K (LY294002) to elucidate pathway involvement. Results: Using G-LISA we determined that stimulation of primary chondrocytes with TGF? activates RhoA. Reciprocally, inhibition of RhoA/ROCK but not other signalling pathways prevents modification of the actin cytoskeleton in responseto TGF?. Inhibition of MEK/ERKsignaling rescued suppression of anabolic gene expression by TGF? including SOX9 mRNA and protein levels. Inhibition of MEK/ERK, Rho/ROCK, p38 MAPK and PI3K signalling pathways differentially controlled the induction of MMP13 and TNF? gene expression. TGF? also induced expression of CCL2 specifically through MEK/ERK activation. In turn, CCL2 treatment induced the expression of MMP3 and TNF?. Finally, we assessed cartilage degradation by immunohistochemical detection of type II collagen cleavage fragments generated by MMPs. Blockade of RhoA/ROCK and MEK/ERK signalling pathways reduced the generation of type IIcollagen cleavage fragments in response to TGF? stimulation. Conclusions: Rho/ROCK signalling mediates TGF?-induced changes inchondrocyte morphology, while MEK/ERK signalling mediates the suppression ofSox9 and its target genes, and CCL2 expression. CCL2, in turn, induces the expression of MMP3 and TNF?, two potent catabolic factors known to be involved in OA. These pathways may represent strategic targets for interventional approaches to treating cartilage degeneration in osteoarthritis. References: 1. Appleton CTG et al. Arthritis Rheum 2007;56:1854-68. 2. Appleton CTG et al. Arthritis Rheum 2007; 56:3693-705.

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