Gene transfer of metalloproteinase transin induces aberrant behavior of cultured mesangial cells

American Journal of Physiology-Renal Physiology

2000

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To understand how isolation and explantation of glomeruli affect the function of resident cells, the present study investigated the transcriptional profile of explanted normal glomeruli. We found that ex vivo incubation of glomeruli spontaneously expressed monocyte chemoattractant protein-1 (MCP-1) and stromelysin, the genes regulated by activator protein-1 (AP-1). The expression was suppressed by heparin and quercetin, the drugs with anti-AP-1 activities. The gene expression was preceded by 1) induction of AP-1 components c-fos and c-jun and 2) phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein (MAP) kinase, and c-Jun NH(2)-terminal kinase (JNK), the upstream inducers/activators of AP-1. Suppression of ERK by PD098059 abrogated induction of c-fos and c-jun, and the p38 MAP kinase inhibitor SB203580 attenuated c-fos expression. Furthermore, treatment with either PD098059, SB203580, or the JNK-AP-1 inhibitor curcumin diminished the expression of MCP-1 and stromelysin. The transcriptional profile of glomerular cells thus alters dramatically after explantation of glomeruli. It is, at least in part, due to activation of multiple MAP kinases that lead to induction of AP-1-dependent gene expression.

mentioning

confidence: 99%

Spontaneous shift in transcriptional profile of explanted glomeruli via activation of the MAP kinase family

Ishikawa

Konta

American Journal of Physiology-Renal Physiology

2000

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“…Using techniques for three-dimensional culture, we previously reported that surrounding ECM elicits differentiation of mesangial cells in vitro [20, 21, 22]. A gene transfection study showed that mesangial cells with aberrant matrix-degrading activity exhibited an activated phenotype in ECM [23]. This line of evidence suggests that the mesangial matrix controls the behavior of mesangial cells and maintains their differentiation in the glomerulus.…”

Section: Tgf-β As An Endogenous Defender Against Glomerular Injurymentioning

confidence: 68%

Evidence for TGF-β-Mediated ‘Defense’ of the Glomerulus: A Blackguard Molecule Rehabilitated?

Fine²

1998

Nephron Exp Nephrol

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Transforming growth factor beta (TGF-β) has been regarded as a ‘blackguard molecule’ that induces glomerular diseases. During the process of glomerulonephritis, upregulated TGF-β stimulates the production of extracellular matrix and inhibits its degradation, leading to excessive matrix deposition. On the other hand, TGF-β has the potential to be anti-inflammatory via inhibition of mitogenesis and production of inflammatory mediators by glomerular cells. This molecule strongly inactivates infiltrating cells, especially macrophages, which play a pivotal role in the generation of glomerular injury. The aim of this article is to summarize the potentially beneficial action of TGF-β in the glomerulus and to address its ‘bright side’ in glomerular inflammation.

“…A cell clone SM43 was established by a limiting dilutional method and identified as being of mesangial cell phenotype as described (25). By using a modified calcium phosphate coprecipitation method (26), SM43 mesangial cells were cotransfected with a regulator plasmid pUHD15-1 encoding tTA (21) and a plasmid pRc/CMV (Invitrogen) that introduces a neomycin phosphotransferase gene. Stable transfectants were selected in the presence of neomycin analogue G418 (750 ,ug/ml; Sigma).…”

Section: Methodsmentioning

confidence: 99%

Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.

Proc. Natl. Acad. Sci. U.S.A.

1996

Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affilnity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a 18-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no .8-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of f8-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of 8-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, 18-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.The renal glomerulus is a microscopic vascular structure scattered throughout the cortex of the kidney. During the past decade, numerous studies have been done to elucidate molecular mechanisms of glomerular diseases. Among various approaches utilized, in vivo gene transfer technology would be one of the most powerful and promising strategies, since this allows us to modulate activity of a certain molecule within the glomerulus (1, 2). However, recent investigations have encountered some hurdles. Because of the microscopic size (100 jam in diameter) and number (3 x 104 to 1 X 106/kidney) of glomeruli, it is difficult to efficiently and site-specifically deliver foreign genes into the majority of the glomeruli. Conventional cationic liposomes or adenoviral vectors, the most popular in vivo gene transfer vectors, were found to be inefficient for this purpose (3,4). A modified liposome system could be useful (5), but exogenous gene expression has been limited to several days (6). To overcome these problems, I established a novel gene transfer system that specifically targets the glomerulus (7-9). In this method, the glomerular mesangial cell ...