ERK, p38, and Smad signaling pathways differentially regulate transforming growth factor-beta1 autoinduction in proximal tubular epithelial cells

Abstract

Transforming growth factor (TGF)-beta1 is a mediator of the final common pathway of fibrosis associated with progressive renal disease, a process in which proximal tubular cells (PTCs) are known to play an important part. The aim of the current study was to examine the mechanism of PTC TGF-beta1 autoinduction. The addition of TGF-beta1 led to increased amounts of TGF-beta1 mRNA and increased de novo protein synthesis. The addition of TGF-beta1 led to increased phosphorylation of R-Smads and activation of extracellular signal-regulated kinase mitogen-activated protein (MAP) kinase and p38 MAP kinase pathways. Use of a dominant-negative Smad3 (Smad3 DN) expression vector, Smad3 small interfering RNA, and inhibition of extracellular signal-regulated kinase and p38 MAP kinase pathways with the chemical inhibitors PD98059 or SB203580 suggested that activation of these signaling pathways occurred independently. Smad3 DN expression, Smad3 small interfering RNA, or the addition of PD98059 inhibited TGF-beta1-dependent stimulation of TGF-beta1 mRNA. Furthermore, Smad3 blockade specifically inhibited activation of the transcription factor AP-1 by TGF-beta1, whereas PD98059 prevented TGF-beta1-dependent nuclear factor-kappaB activation. In contrast inhibition of p38 MAP kinase inhibited de novo TGF-beta1 protein synthesis but did not influence TGF-beta1 mRNA expression or activation of either transcription factor. In summary, in PTCs, TGF-beta1 autoinduction requires the coordinated action of independently regulated Smad and non-Smad pathways. Furthermore these pathways regulate distinct transcriptional and translational components of TGF-beta1 synthesis.

FIGURE 1

TGF-β1 increases TGF-β1 mRNA expression. Confluent monolayers of HK-2 cells were growth-arrested and exposed to recombinant TGF-β1 (0 to 2 ng/ml) for up to 48 hours. A: After RNA isolation TGF-β1 mRNA was detected as a single 2.4-kb transcript by Northern analysis. Equal mRNA loading was confirmed by stripping the blot and reprobing for GAPDH. In parallel experiments cells were stimulated by the addition of TGF-β1 (1 ng/ml) for 24 hours. B: After RNA isolation and reverse transcription, TGF-β1 mRNA was quantified by quantitative PCR. Data represent mean ± SD, n = 8.

FIGURE 2

Determination of translational efficiency and de novo protein synthesis. Confluent monolayers of HK-2 cells were growth-arrested and exposed to TGF-β1 (1 ng/ml) for 12 hours before extraction of the cytosol and separation of mRNA on a sucrose gradient as described in Materials and Methods. A: Subsequently, a Northern blot of the fractionated mRNA was probed for TGF-β1. Fractions 1 to 11 represent tRNAs, free ribosomal subunits, monosomes, and untranslated mRNAs in the form of messenger ribonucleoprotein particles. Fractions 12 to 22 represent polysomes of increasing size. B: Graphical representation of the polysome distribution of TGF-β1 mRNA, expressed as percentage of the total TGF-β1 mRNA detected on a given blot in each fraction, with control shown as shaded area and TGF-β1-stimulated as the unshaded area. C: Incorporation of radioactive amino acids into newly synthesized protein by metabolic labeling was used to assess de novo TGF-β1 synthesis. Cells were stimulated with TGF-β1 (0 to 5 ng/ml) in the presence of 40 μCi of ³H-radiolabeled amino acid mixture (1000 μCi/ml; Amersham). Supernatant samples were subsequently collected for TGF-β1 immunoprecipitation, and radiolabeled TGF-β1 was detected by autoradiography.

FIGURE 3

TGF-β1 mediated activation of signaling pathways. Confluent monolayers of HK2 cells were stimulated by the addition of TGF-β1 (1 ng/ml) for up to 6 hours. At the time points indicated, total cell extracts were generated, and immunoblot analysis of lysate samples for phosphorylated-Smad/total Smad (A), phosphorylated ERK/total-ERK (B), and phosphorylated p38/total p38 (C) was performed.

FIGURE 4

Smad activation occurs independently of MAP kinase activation. To determine the role of ERK and p38 MAP kinase in TGF-β1-mediated Smad activation, cells were stimulated with TGF-β1 (1 ng/ml) either alone or in the presence of PD98059 (A) or SB203580 (B) for 30 minutes. Subsequently, total cell extracts were generated and immunoblot analysis of lysate samples for phosphorylated Smad/total Smad.

FIGURE 5

MAP kinase activation occurs after expression of Smad dominant-negative expression vector. Inhibition of Smad3 activation was achieved by transient transfection with a c-myc-tagged dominant-negative Smad3 expression vector. A: Transfection efficiency of the dominant-negative Smad3 expression vector. Histogram of FL-1 fluorescence in EGFP-transfected versus mock-transfected cells. Transfection efficiency, based on the proportion of cells in gated region M1, is estimated at 55%. Efficacy of the expression vector was confirmed by co-transfection of 1.0 μg of the dominant-negative (DN) expression vector or empty vector (EV) together with 0.9 μg of the Smad-responsive (SBE)₄-Lux reporter and 0.1 μg of the Renilla luciferase construct using 6 μl of the mixed lipofection reagent FuGene6. B: Twenty-four hours after transfection cells were stimulated with 1 ng/ml TGF-β1 for 6 hours before quantitation of luciferase content. Results are expressed as ratios of firefly/Renilla luciferase and represent mean ± SD, n = 3. In parallel experiments, cells were transiently transfected with 1 μg of the c-myc-tagged dominant-negative Smad3 expression vector using 3 μl of FuGene6. Twenty-four hours after transfection, cells were stimulated with TGF-β1 (1 ng/ml) for 30 minutes. Subsequently, total cell extracts were generated, and immunoblot analysis of lysate samples for ERK/total-ERK (C) and phosphorylated p38/total p38 (D) was performed. E: In parallel experiments, overexpression of the vector was confirmed by c-myc immunoblot.

FIGURE 6

MAP kinase activation occurs after gene silencing of Smad3 by Smad3 siRNA. Cells were transfected with Smad3 siRNA for 48 hours before cell lysis by the addition of Trireagent. A: Smad3 mRNA was subsequently analyzed by quantitative PCR. In parallel experiments, cells transfected with Smad3 siRNA were stimulated with TGF-β1 (1 ng/ml) for 30 minutes. B: Subsequently, total cell extracts were generated and immunoblot analysis of lysate samples for ERK/total-ERK and phosphorylated p38/total p38 was performed.

FIGURE 7

Inhibition of ERK and Smad3, but not p38, inhibits autocrine TGF-β1 transcription. HK2 cell monolayers were stimulated with TGF-β1 (1 ng/ml) for 24 hours either alone or in the presence of either PD98059 or SB203580 at the concentrations indicated. After RNA isolation and reverse transcription, TGF-β1 mRNA was quantified by quantitative PCR. Data represent mean ± SD, n = 4. Inhibition of Smad3 was achieved by transient transfection of the Smad3 dominant-negative expression vector (A) or by gene silencing using Smad3 siRNA (B). Cells were transiently transfected with either the c-myc-tagged dominant-negative Smad3 expression vector or Smad3 siRNA. Twenty-four hours after transfection, cells were stimulated with TGF-β1 (1 ng/ml) for a further 24 hours, and TGF-β1 mRNA was quantified by quantitative PCR. Data represent mean ± SD, n = 4.

FIGURE 8

Transcription factor activation. Mobility shift experiments were performed with nuclear extracts from HK-2 cells cultured for up to 6 hours in the presence of TGF-β1 (1 ng/ml). Nuclear extract was incubated with probes for AP-1 (A–C) and NF-κB (D, E). Identification of proteins involved in AP-1 activation was determined by mobility shift analysis with antibodies to c-fos/c-jun (B) and NF-κB activation using antibodies to p50, p65, cRel, RelB, or p52 as indicated (E). The role of Smad3 in either AP-1 (C) or NF-κB (F) activation was examined by TGF-β1 (1 ng/ml) stimulation of HK2 cells transiently transfected with Smad3 dominant-negative expression vector (DN) for 24 hours before performing the mobility shift assay. In the control experiment, cells transiently transfected with the empty vector (EV) were stimulated with TGF-β1. Likewise, the role of the ERK MAP kinase and p38 MAP kinase pathways in AP-1 activation was examined by TGF-β1 (1 ng/ml) stimulation of HK2 cells for 10 minutes in the presence of either 8 μmol/L PD98059 (PD) or 0.8 μmol/L SB203580 (SB) before analysis by mobility shift assay (C), and their role in NF-κB activation was similarly examined in cells exposed to TGF-β1 for 6 hours (F).

FIGURE 9

Inhibition of ERK MAP kinase and P38 MAP kinase inhibits TGF-β1-stimulated TGF-β1 de novo protein synthesis. A: HK-2 cells were stimulated with TGF-β1 (1 ng/ml) either alone or in combination with PD98059 (8 μmol/L) or SB203580 (0.8 μmol/L) for 48 hours. B: In parallel experiments, to demonstrate specificity of the kinase inhibitors used, cells were stimulated with TGF-β1 in combination with the JNK MAP kinase inhibitor recombinant L-JNKI1 (JNKi) at a concentration of 10 μmol/L. All experiments were performed in the presence of 40 μCi of ³H-radiolabeled amino acid mixture (1000 μCi/ml; Amersham). Supernatant samples were subsequently collected for TGF-β1 immunoprecipitation and radiolabeled TGF-β1 was detected by autoradiography.