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The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells.
Kidney Int. 1999 Oct; 56(4):1354-65.KI

Abstract

BACKGROUND

Transforming growth factor-beta (TGF-beta) signals through a unique set of intracellular proteins, called SMADs, that have been characterized mainly in transient overexpression systems. Because several models of glomerulosclerosis suggest a role for TGF-beta in the extracellular matrix accumulation, we sought to characterize the role of SMAD proteins in mediating TGF-beta1 responses in a more physiological system using nontransformed human mesangial cells.

METHODS

Endogenous SMAD expression and its modulation by TGF-beta1 were evaluated by Western and Northern blot analyses. Phosphorylation of Smad2 and Smad3 was determined by both phospholabeling and immunoblot. SMAD function and its role in type I collagen transcription were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs.

RESULTS

Cultured human mesangial cells express Smad2, Smad3, and Smad4 proteins. TGF-beta1 down-regulated Smad3 mRNA and protein expression, respectively, after 4 and 24 hours of treatment, whereas Smad2 and Smad4 were less affected. Both Smad2 and Smad3 were phosphorylated in response to TGF-beta1 beginning at 5 minutes, with maximal phosphorylation at 15 minutes, and decreasing phosphorylation by 2 hours. Smad2/3 and Smad4 coimmunoprecipitate only after TGF-beta1 treatment. The activity of a transiently transfected, TGF-beta-responsive construct, p3TP-Lux, was stimulated 3.6-fold by TGF-beta1. Overexpressed wild-type Smad3 increased basal luciferase activity, which was further stimulated by TGF-beta1. A dominant negative mutant form of Smad3 lacking the C-terminal serine phosphoacceptor sites (Smad3A) inhibited TGF-beta1-induced luciferase activity. TGF-beta1 also increased the activation of an alpha2(I) collagen promoter-luciferase reporter construct transfected into mesangial cells. This activation was inhibited by cotransfection with the Smad3A mutant.

CONCLUSIONS

Smad2, Smad3, and Smad4 are present and activated by TGF-beta1 in human mesangial cells. The SMAD pathway is functional in these cells and appears to be involved in TGF-beta1-induced type I collagen gene transcription. These findings raise the possibility that SMAD signaling plays a role in glomerular matrix accumulation.

Authors+Show Affiliations

Department of Pediatrics and Children's Memorial Institute for Education and Research, Northwestern University Medical School, Chicago, Illinois, USA. anne-c@nwu.eduNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10504488

Citation

Poncelet, A C., et al. "The Transforming Growth factor-beta/SMAD Signaling Pathway Is Present and Functional in Human Mesangial Cells." Kidney International, vol. 56, no. 4, 1999, pp. 1354-65.
Poncelet AC, de Caestecker MP, Schnaper HW. The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells. Kidney Int. 1999;56(4):1354-65.
Poncelet, A. C., de Caestecker, M. P., & Schnaper, H. W. (1999). The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells. Kidney International, 56(4), 1354-65.
Poncelet AC, de Caestecker MP, Schnaper HW. The Transforming Growth factor-beta/SMAD Signaling Pathway Is Present and Functional in Human Mesangial Cells. Kidney Int. 1999;56(4):1354-65. PubMed PMID: 10504488.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells. AU - Poncelet,A C, AU - de Caestecker,M P, AU - Schnaper,H W, PY - 1999/10/3/pubmed PY - 1999/10/3/medline PY - 1999/10/3/entrez SP - 1354 EP - 65 JF - Kidney international JO - Kidney Int VL - 56 IS - 4 N2 - BACKGROUND: Transforming growth factor-beta (TGF-beta) signals through a unique set of intracellular proteins, called SMADs, that have been characterized mainly in transient overexpression systems. Because several models of glomerulosclerosis suggest a role for TGF-beta in the extracellular matrix accumulation, we sought to characterize the role of SMAD proteins in mediating TGF-beta1 responses in a more physiological system using nontransformed human mesangial cells. METHODS: Endogenous SMAD expression and its modulation by TGF-beta1 were evaluated by Western and Northern blot analyses. Phosphorylation of Smad2 and Smad3 was determined by both phospholabeling and immunoblot. SMAD function and its role in type I collagen transcription were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. RESULTS: Cultured human mesangial cells express Smad2, Smad3, and Smad4 proteins. TGF-beta1 down-regulated Smad3 mRNA and protein expression, respectively, after 4 and 24 hours of treatment, whereas Smad2 and Smad4 were less affected. Both Smad2 and Smad3 were phosphorylated in response to TGF-beta1 beginning at 5 minutes, with maximal phosphorylation at 15 minutes, and decreasing phosphorylation by 2 hours. Smad2/3 and Smad4 coimmunoprecipitate only after TGF-beta1 treatment. The activity of a transiently transfected, TGF-beta-responsive construct, p3TP-Lux, was stimulated 3.6-fold by TGF-beta1. Overexpressed wild-type Smad3 increased basal luciferase activity, which was further stimulated by TGF-beta1. A dominant negative mutant form of Smad3 lacking the C-terminal serine phosphoacceptor sites (Smad3A) inhibited TGF-beta1-induced luciferase activity. TGF-beta1 also increased the activation of an alpha2(I) collagen promoter-luciferase reporter construct transfected into mesangial cells. This activation was inhibited by cotransfection with the Smad3A mutant. CONCLUSIONS: Smad2, Smad3, and Smad4 are present and activated by TGF-beta1 in human mesangial cells. The SMAD pathway is functional in these cells and appears to be involved in TGF-beta1-induced type I collagen gene transcription. These findings raise the possibility that SMAD signaling plays a role in glomerular matrix accumulation. SN - 0085-2538 UR - https://www.unboundmedicine.com/medline/citation/10504488/The_transforming_growth_factor_beta/SMAD_signaling_pathway_is_present_and_functional_in_human_mesangial_cells_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0085-2538(15)46445-2 DB - PRIME DP - Unbound Medicine ER -