Targeted disruption of Smad3 reveals an essential role in transforming growth factor beta-mediated signal transduction

Abstract

The Smads are a family of nine related proteins which function as signaling intermediates for the transforming growth factor beta (TGF-beta) superfamily of ligands. To discern the in vivo functions of one of these Smads, Smad3, we generated mice harboring a targeted disruption of this gene. Smad3 null mice, although smaller than wild-type littermates, are viable, survive to adulthood, and exhibit an early phenotype of forelimb malformation. To study the cellular functions of Smad3, we generated Smad3 null mouse embryonic fibroblasts (MEFs) and dermal fibroblasts. We demonstrate that null MEFs have lost the ability to form Smad-containing DNA binding complexes and are unable to induce transcription from the TGF-beta-responsive promoter construct, p3TP-lux. Using the primary dermal fibroblasts, we also demonstrate that Smad3 is integral for induction of endogenous plasminogen activator inhibitor 1. We subsequently demonstrate that Smad3 null MEFs are partially resistant to TGF-beta's antiproliferative effect, thus firmly establishing a role for Smad3 in TGF-beta-mediated growth inhibition. We next examined cells in which Smad3 is most highly expressed, specifically cells of immune origin. Although no specific developmental defect was detected in the immune system of the Smad3 null mice, a functional defect was observed in the ability of TGF-beta to inhibit the proliferation of splenocytes activated by specific stimuli. In addition, primary splenocytes display defects in TGF-beta-mediated repression of cytokine production. These data, taken together, establish a role for Smad3 in mediating the antiproliferative effects of TGF-beta and implicate Smad3 as a potential effector for TGF-beta in modulating immune system function.

FIG. 1

Targeted disruption of Smad3. (A) Smad3 genomic structure and targeting strategy. The Smad3 genomic clone used for the creation of the targeting vector is diagrammed. The black box denotes the first exon of Smad3. ATG denotes the initiating methionine. The first exon coding sequence was replaced by a neomycin expression cassette (NEO), creating an EcoRI digest size difference between the wild-type and targeted loci. Ep denotes the EcoRI-HindIII DNA fragment used as a probe for Southern blotting. P1, P2, and P3 denote the locations of primers used for PCR screening. Restriction sites are abbreviated as follows: R, EcoRI; H, HindIII; E, EheI; B, BamHI. HSV-TK, herpes simplex virus thymidine kinase. (B) Southern and PCR detection of the targeted allele. The targeted allele can be distinguished from the wild type (W.T.) both by Southern blotting of EcoRI-digested genomic DNA with the Ep probe and by PCR using the primers indicated in panel A. (C) Northern blot analysis of Smad3 and Smad4. Northern analyses were performed on RNA derived from multiple tissues of an adult (2-month-old) C57BL/6 mouse and a 3′ untranslated sequence probe for Smad3 and a coding-sequence probe for Smad4. Organs are abbreviated as follows: B, brain; H, heart; M, skeletal muscle; I, small intestine; Lu, lung; K, kidney; T, thymus; S, spleen. (D) Western blot analysis of Smad3 expression. The top panel shows Western analysis using an antibody created against a peptide in the central linker domain of Smad3 on thymic protein extract from wild-type, heterozygous, and knockout mice. The bottom two panels demonstrate the specificity of this antibody among overexpressed Smad family members. HA-tagged Smad1, Smad2, and Smad4 and Flag-tagged Smad3 were overexpressed in COS cells from which protein extract were isolated and used for Western analysis. Identical blots were probed with the Smad3-specific antibody (middle panel) and a mixture of αHA and αFlag (bottom panel). In all panels of all figures, +/+ denotes Smad3 wild type, +/− denotes Smad3 heterozygous, and −/− denotes Smad3 null.

FIG. 2

Smad3 null mice are smaller than wild-type littermates and have an incompletely penetrant forepaw defect. (A) Mouse weights over time (days) in a single, representative litter. (B) Picture of the torqued-wrist defect (arrow) in a 14-day-old null mouse. (C) The skin was removed from the forelimbs of a 14-day-old Smad3 null mouse (left) and a wild-type littermate (right) to better show the severe bending of the forepaw wrist joint of the Smad3 null mouse.

FIG. 3

Smad3 is required for TGF-β-mediated growth inhibition in MEFs. (A) Primary MEFs were created from embryonic day 14 mice. Western blotting for Smad3 was performed to determine if these MEFs express Smad3. (B) Smad3 is required for TGF-β-mediated growth inhibition in primary MEFs. MEFs were assayed for TGF-β-mediated growth inhibition after 24 and 48 h of treatment by measurement of [³H]thymidine incorporation. Bars represent the average thymidine incorporation for triplicate wells for each growth condition. (C) TGF-β-mediated growth inhibition in these MEFs is cell autonomous. Various proportions of wild-type (WT) and knockout (KO) MEFs were seeded into single wells as indicated below the bars. Thymidine incorporation assays were performed as for panel B. Data are presented as percent growth inhibition or percent reduction in thymidine incorporation upon TGF-β treatment.

FIG. 4

Smad3 is required for TGF-β-mediated Smad-containing DNA binding complex formation and activation of 3TP-Lux in primary MEFs and for TGF-β-mediated induction of the PAI-1 gene in primary dermal fibroblasts. (A) Loss of a Smad-containing DNA binding complex in the Smad3 null MEFS. EMSAs were performed with nuclear extract from MEFs of the indicated genotype, either treated with TGF-β for 30 min or untreated, and a probe derived from the TGF-β-responsive region of the promoter-reporter construct, p3TP-lux. The arrow indicates the TGF-β-inducible DNA binding complex. (B) Smad3 is required for induction of the p3TP-lux reporter construct. The indicated DNAs were transfected into MEFs of the indicated genotype. Twelve hours after transfection, the cells were treated with 100 pM TGF-β for an additional 24 h, and TGF-β-induced luciferase activity (relative luciferase units [RLU]) from this reporter construct was assayed. Bars represent the average luciferase activity of duplicate transfections in a single experiment; error bars represent the standard deviation. Fold induction by TGF-β is indicated over each set of bars. (C) Smad3 is an integral component of the TGF-β-mediated induction of the endogenous PAI-1 gene. Smad3 heterozygote and null primary dermal fibroblasts were treated with TGF-β for 8 h. The arrow represents [³⁵S]methionine-labeled, extracellular matrix-associated PAI-1, assayed as described in Materials and Methods.

FIG. 5

Assay of TGF-β’s effects in primary splenocytes reveals both Smad3-dependent and Smad3-independent growth-inhibitory signaling pathways. (A) Smad3 is not required for TGF-β-mediated growth inhibition in primary unstimulated splenocytes. Primary splenocytes were isolated from 8-week-old mice and cultured in the presence or absence of 100 pM TGF-β for 48 h. Cells were incubated with [³H]thymidine for the last 4 h of culture, after which the splenocytes were harvested and ³H incorporation was measured. Bars indicate the average of three identically treated wells for each growth condition; error bars represent the standard deviation. (B) Smad3 is required for TGF-β-mediated growth inhibition of αCD3-stimulated splenocytes. Primary splenocytes were isolated from 8-week-old mice and cultured in the presence of the indicated growth stimuli in the presence or absence of 100 pM TGF-β. Cellular proliferation was assayed by [³H]thymidine incorporation as for panel A. (C) Smad3 is expressed in both B and T cells. Western blotting for Smad3 was performed on purified B and T cells from mature wild-type spleens.

FIG. 6

TGF-β-mediated growth inhibition of αCD3-stimulated splenocytes is associated with a decrease in G₁ Cdk activity and cytokine expression. (A) Splenocytes were harvested from wild-type and knockout mice and cultured with αCD3 in the presence or absence of TGF-β. Cell lysates were prepared and subjected to Western blotting for cyclin E, Cdk2, p27, and Rb (lower panels). In addition, Cdk2 kinase activity was assayed by immunoprecipitation of Cdk2 and evaluation of its ability to phosphorylate the exogenous substrate, histone H1 (top panel). (B) Cytokine production was assayed on splenocytes from wild-type and Smad3 null mice treated as for panel A, using an RNase protection assay. The identity of each band is indicated on the right. L32 and GAPDH are controls for mRNA quantity and quality. (C) The intensity of the cytokine RPA bands in panel B was determined by densitometry. Plotted are the relative intensities of each band, with wild-type levels of each cytokine set at 100%.

FIG. 7

FACs analyses of thymocytes and splenocytes isolated from wild-type and Smad3 null mice demonstrate normal T-cell and B-cell development. (A and B) Representative FACs analysis of wild-type and Smad3 null thymocytes, using αCD4-PE and αCD8-FITC. (C to F) Representative FACS analysis of wild-type and Smad3 null splenocytes, using the indicated conjugated antibodies. All data was gated for viable cells by the absence of 7AAD staining. Percentages represent the proportions of viable cells in each region or quadrant.