Constitutive expression of endothelin gene in cultured human mesangial cells and its modulation by transforming growth factor-beta, thrombin, and a thromboxane A2 analogue.
The present study was designed to assess whether human glomerular mesangial cells in culture express preproendothelin gene and whether endothelin gene expression in the mesangium is regulated by factors potentially released by inflammatory cells and platelets infiltrating the glomerular tuft during the course of various types of glomerulonephritis. For this purpose mesangial cells were incubated for 6 hours in the presence of absence of interleukin 1 beta (IL-1 beta), transforming growth factor-beta (TBF-beta), the thromboxane A2 analogue U-46619, and thrombin. Resting mesangial cells expressed a 2.3-kilobase mRNA on blot hybridization analysis with a human cDNA preproendothelin probe, indicating that this type of cells, in addition to glomerular endothelial cells, constitutively expresses endothelin gene. IL-1 beta did not change endothelin mRNA levels in respect to unstimulated mesangial cells. At variance, TGF-beta, U-46619, and thrombin had a marked effect on endothelin mRNA, stimulating a 3- to 8-fold increase over basal levels. Quantification of actin mRNA and analysis of the autoradiographic signals provided validation of the difference in the endothelin mRNA levels. Expression of preproendothelin mRNA in either resting or stimulated mesangial cells was associated with synthesis and release of the corresponding peptide in the cell supernatant as determined by a specific radioimmunoassay for endothelin. Endothelin production from IL-1 beta stimulated mesangial cells was not different from that of unstimulated cells, whereas a significant (p less than 0.01) increase in endothelin production was observed after cell stimulation with TGF-beta, U-46619, and thrombin. The demonstration that mesangial cells constitutively express mRNA for preproendothelin and release endothelin into culture medium, together with the finding that endothelin gene expression and production in mesangial cells are regulated by molecules potentially released at glomerular level during an inflammatory reaction may suggest that endothelin participates in the complex process of glomerular disease progression.
Mario Negri Institute for Pharmacological Research, Bergamo, Italy.
C
C
Zoja
S
S
Orisio
N
N
Perico
A
A
Benigni
M
M
Morigi
L
L
Benatti
A
A
Rambaldi
G
G
Remuzzi
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D001345:Autoradiography; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D011863:Radioimmunoassay; D013917:Thrombin / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000494:pharmacology*; D015291:Transforming Growth Factors / Q000494:pharmacology*
D019332:Endothelin-1; D016232:Endothelins; D007375:Interleukin-1; D011450:Prostaglandin Endoperoxides, Synthetic; D011498:Protein Precursors; D012333:RNA, Messenger; D013928:Thromboxane A2; D012313:RNA; D015291:Transforming Growth Factors; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D004247:DNA; D013917:Thrombin