Joshua P. Frederick

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 β (TGF-β) 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-β-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-β’s antiproliferative effect, thus firmly establishing a role for Smad3 in TGF-β-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-β to inhibit the proliferation of splenocytes activated by specific stimuli. In addition, primary splenocytes display defects in TGF-β-mediated repression of cytokine production. These data, taken together, establish a role for Smad3 in mediating the antiproliferative effects of TGF-β and implicate Smad3 as a potential effector for TGF-β in modulating immune system function.

Tumor suppressor Smad4 is a transforming growth factor beta-inducible DNA binding protein.

Members of the Smad family of proteins are thought to play important roles in transforming growth factor beta (TGF-beta)-mediated signal transduction. In response to TGF-beta, specific Smads become inducibly phosphorylated, form heteromers with Smad4, and undergo nuclear accumulation. In addition, overexpression of specific Smad combinations can mimic the transcriptional effect of TGF-beta on both the plasminogen activator inhibitor 1 (PAI-1) promoter and the reporter construct p3TP-Lux. Although these data suggest a role for Smads in regulating transcription, the precise nuclear function of these heteromeric Smad complexes remains largely unknown. Here we show that in Mv1Lu cells Smad3 and Smad4 form a TGF-beta-induced, phosphorylation-dependent, DNA binding complex that specifically recognizes a bipartite binding site within p3TP-Lux. Furthermore, we demonstrate that Smad4 itself is a DNA binding protein which recognizes the same sequence. Interestingly, mutations which eliminate the Smad DNA binding site do not interfere with either TGF-beta-dependent transcriptional activation or activation by Smad3/Smad4 cooverexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-beta-dependent transcriptional activation and activation in response to Smad3/Smad4 cooverexpression. Furthermore, concatemerized AP1 sites, in isolation, are activated by Smad3/Smad4 cooverexpression and, to a certain extent, by TGF-beta. Taken together, these data suggest that the Smad3/Smad4 complex has at least two separable nuclear functions: it forms a rapid, yet transient sequence-specific DNA binding complex, and it potentiates AP1-dependent transcriptional activation.

Smad3-Smad4 and AP-1 Complexes Synergize in Transcriptional Activation of the c-Jun Promoter by Transforming Growth Factor β

ABSTRACT Transcriptional regulation by transforming growth factor β (TGF-β) is a complex process which is likely to involve cross talk between different DNA responsive elements and transcription factors to achieve maximal promoter activation and specificity. Here, we describe a concurrent requirement for two discrete responsive elements in the regulation of the c-Jun promoter, one a binding site for a Smad3-Smad4 complex and the other an AP-1 binding site. The two elements are located 120 bp apart in the proximal c-Jun promoter, and each was able to independently bind its corresponding transcription factor complex. The effects of independently mutating each of these elements were nonadditive; disruption of either sequence resulted in complete or severe reductions in TGF-β responsiveness. This simultaneous requirement for two distinct and independent DNA binding elements suggests that Smad and AP-1 complexes function synergistically to mediate TGF-β-induced transcriptional activation of the c-Jun promoter.

Transforming Growth Factor β-Mediated Transcriptional Repression of c-myc Is Dependent on Direct Binding of Smad3 to a Novel Repressive Smad Binding Element

ABSTRACT Smad proteins are the most well-characterized intracellular effectors of the transforming growth factor β (TGF-β) signal. The ability of the Smads to act as transcriptional activators via TGF-β-induced recruitment to Smad binding elements (SBE) within the promoters of TGF-β target genes has been firmly established. However, the elucidation of the molecular mechanisms involved in TGF-β-mediated transcriptional repression are only recently being uncovered. The proto-oncogene c-myc is repressed by TGF-β, and this repression is required for the manifestation of the TGF-β cytostatic program in specific cell types. We have shown that Smad3 is required for both TGF-β-induced repression of c-myc and subsequent growth arrest in keratinocytes. The transcriptional repression of c-myc is dependent on direct Smad3 binding to a novel Smad binding site, termed a repressive Smad binding element (RSBE), within the TGF-β inhibitory element (TIE) of the c-myc promoter. The c-myc TIE is a composite element, comprised of an overlapping RSBE and a consensus E2F site, that is capable of binding at least Smad3, Smad4, E2F-4, and p107. The RSBE is distinct from the previously defined SBE and may partially dictate, in conjunction with the promoter context of the overlapping E2F site, whether the Smad3-containing complex actively represses, as opposed to transactivates, the c-myc promoter.

Impaired Immune Responses and B-Cell Proliferation in Mice Lacking the Id3 Gene

ABSTRACT B-lymphocyte activation and proliferation induced by the B-cell receptor (BCR) signals are important steps in the initiation of humoral immune responses. How the BCR signals are translated by nuclear transcription factors into cell cycle progression is poorly understood.Id3 is an immediate-early gene responding to growth and mitogenic signals in many cell types including B cells. The primary function of the Id3 protein has been defined as that of inhibitor of basic-helix-loop-helix (bHLH) transcription factors. The interaction between Id3 and bHLH proteins, many of which are essential for cellular differentiation, has been proposed as a key regulatory event leading to cellular proliferation instead of differentiation. To further investigate the role of Id3 in tissue and embryo development and the mechanism of Id3-mediated growth regulation, we generated and analyzedId3-deficient mice. While these mice display no overt abnormality in tissue and embryo development, their humoral immunity is compromised. The amounts of immunoglobulins produced inId3-deficient mice immunized with a T-cell-dependent antigen and a type 2 T-cell-independent antigen are attenuated and severely impaired, respectively. Further analysis of lymphocytes isolated from Id3-deficient mice reveals a B-cell defect in their proliferation response to BCR cross-linking but not to lipopolysaccharide or a combination of BCR cross-linking and interleukin-4. Analyses of cultured lymphocytes also suggest involvement of Id3 in cytokine production in T cells and isotype switching in B cells. Finally, the proliferation defect inId3-deficient B cells can be rescued by ectopic expression of Id1, a homologue of Id3. Taken together, these results define a necessary and specific role for Id3 in mediating signals from BCR to cell cycle progression during humoral immune responses.

The Loss of Smad3 Results in a Lower Rate of Bone Formation and Osteopenia Through Dysregulation of Osteoblast Differentiation and Apoptosis

Smad3 is a well‐characterized intracellular effector of the transforming growth factor β (TGF‐β) signaling pathway and was implicated recently in the potentiation of vitamin D receptor (VDR)‐mediated signaling. Given that both TGF‐β and vitamin D are important regulators of bone remodeling, it is expected that Smad3 plays an integral role in normal maintenance of bone. However, the exact mechanisms by which Smad3 functions in bone remodeling are unknown. Here, we show that mice with targeted deletion of Smad3 are osteopenic with less cortical and cancellous bone compared with wild‐type littermates. Decreases in bone mineral density (BMD) in Smad3 null mice reflect the inability of osteoblasts to balance osteoclast activity, although osteoclast numbers are normal and vitamin D mediated serum calcium homeostasis is maintained. The osteopenia of Smad3 null mice is attributed to a decreased rate of bone formation associated with increased osteocyte number and apoptosis. These findings are supported by studies with isolated primary osteoblasts that show TGF‐β can no longer inhibit the differentiation of osteoblasts in the absence of Smad3; yet, TGF‐β‐stimulated proliferation remains intact. Together these data support a model that a loss of Smad3 increases the osteocyte fate of the osteoblast and decreases the duration of osteoblast function by shortening lifespan, ultimately resulting in osteopenia.

Essential Role for Smad3 in Regulating MCP-1 Expression and Vascular Inflammation

Abstract— Transforming growth factor (TGF)-&bgr;1 is a pleiotropic growth factor with known inhibitory effects on immune cell activation. However, the specific mechanism(s) and in vivo significance of the effectors of TGF-&bgr;1 modulation in the context of vascular inflammation are not well characterized. The chemokine monocyte chemoattractant protein (MCP)-1 is critical for the recruitment of macrophages in inflammatory disease states. In this study, we provide definitive evidence that the ability of TGF-&bgr;1 to inhibit MCP-1 expression is mediated via its effector Smad3. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous MCP-1. Conversely, TGF-&bgr;1 inhibition of cytokine-mediated induction of MCP-1 expression was completely blocked in Smad3-deficient macrophages. Consistent with this impaired response, cardiac allografts in Smad3-deficient mice developed accelerated intimal hyperplasia with increased infiltration of adventitial macrophages expressing MCP-1. Previous studies show that MCP-1 inducibility is regulated by an AP-1 complex composed of c-Jun/c-Fos heterodimers. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on AP-1 DNA-protein binding and activity. Thus, Smad3 plays an essential role in modulating vascular inflammation characteristic of transplant-associated arteriopathy, is important in regulating MCP-1 expression, and plays a critical role in the ability of TGF-&bgr;1 to repress stimuli from a major inflammatory signaling pathway.

A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation

Sulfation is an important biological process that modulates the function of numerous molecules. It is directly mediated by cytosolic and Golgi sulfotransferases, which use 3′-phosphoadenosine 5′-phosphosulfate to produce sulfated acceptors and 3′-phosphoadenosine 5′-phosphate (PAP). Here, we identify a Golgi-resident PAP 3′-phosphatase (gPAPP) and demonstrate that its activity is potently inhibited by lithium in vitro. The inactivation of gPAPP in mice led to neonatal lethality, lung abnormalities resembling atelectasis, and dwarfism characterized by aberrant cartilage morphology. The phenotypic similarities of gPAPP mutant mice to chondrodysplastic models harboring mutations within components of the sulfation pathway lead to the discovery of undersulfated chondroitin in the absence of functional enzyme. Additionally, we observed loss of gPAPP leads to perturbations in the levels of heparan sulfate species in lung tissue and whole embryos. Our data are consistent with a model that clearance of the nucleotide product of sulfotransferases within the Golgi plays an important role in glycosaminoglycan sulfation, provide a unique genetic basis for chondrodysplasia, and define a function for gPAPP in the formation of skeletal elements derived through endochondral ossification.

HUMAN SUBSTANCE P RECEPTOR EXPRESSED IN Sf9 CELLS COUPLES WITH MULTIPLE ENDOGENOUS G PROTEINS

To identify the G proteins involved in the function of human substance P receptor (hSPR), the receptor was expressed in Sf9 cells using the baculovirus expression system. Maximal hSPR expression was up to 65 pmol/mg membrane protein. The following data indicated that hSPR in Sf9 membranes is coupled to endogenous G proteins: 1) binding of agonist radioligand [125I]BHSP to the receptor was sensitive to guanine nucleotides; and 2) stimulation of the receptor increased [35S]GTPgammaS binding. The hSPR-associated G proteins were identified by photoaffinity labeling with [alpha-32P]-azidoanilido GTP ([alpha-32P]AAGTP), followed by immunoprecipitation of the labeled G proteins with antibodies specific for various Galpha-subunits. These experiments showed that stimulation of hSPR in Sf9 membranes activated multiple endogenous G proteins including Galpha(o), Galpha(q/11), and Galpha(s). While hSPRs ability to associate with Gq/11 is well-documented, the present study provides the first evidence of hSPRs potential to activate Galpha(o) and Galpha(s).

Smads “Freeze” When They Ski

The transforming growth factor-beta (TGF-beta) signal is manifest through activated heteromeric Smad complex-mediated transcriptional modulation of target genes that translates into diverse, context-specific biologic effects, such as the potent TGF-beta-initiated cytostatic program. The TGF-beta pathway is effectively antagonized through the direct binding and subsequent repression of activated Smad heteromeric complexes by the Ski family of proto-oncoproteins.