Mark A. Richardson

The MAD-Related Protein Smad7 Associates with the TGFβ Receptor and Functions as an Antagonist of TGFβ Signaling

Abstract TGFβ signaling is initiated when the type I receptor phosphorylates the MAD-related protein, Smad2, on C-terminal serine residues. This leads to Smad2 association with Smad4, translocation to the nucleus, and regulation of transcriptional responses. Here we demonstrate that Smad7 is an inhibitor of TGFβ signaling. Smad7 prevents TGFβ-dependent formation of Smad2/Smad4 complexes and inhibits the nuclear accumulation of Smad2. Smad7 interacts stably with the activated TGFβ type I receptor, thereby blocking the association, phosphorylation, and activation of Smad2. Furthermore, mutations in Smad7 that interfere with receptor binding disrupt its inhibitory activity. These studies thus define a novel function for MAD-related proteins as intracellular antagonists of the type I kinase domain of TGFβ family receptors.

Plasmid cloning vectors that integrate site-specifically in Streptomyces spp.

Cloning vectors based on the Streptomyces ambofaciens plasmid pSAM2 and the streptomycete phage phi C31 were developed for use in Streptomyces spp. These vectors replicate in Escherichia coli but integrate by site-specific recombination in Streptomyces spp. Both pSAM2-based and phi C31-based vectors transformed a number of different Streptomyces spp; however, the phi C31-based vectors consistently transformed at higher frequencies than pSAM2-based vectors. Southern analysis indicated that the phi C31-based vectors integrated at a unique site in the S. ambofaciens chromosome, while the pSAM2-based vectors gave complex patterns which could indicate structural instability or use of multiple loci. Both types of vectors utilize the apramycin (Am)-resistance gene which can be selected in E. coli and Streptomyces spp. with either Am or the commercially available antibiotic Geneticin (G418).

Engineering the proteolytic specificity of activated protein C improves its pharmacological properties

Human activated protein C (APC) is an antithrombotic, antiinflammatory serine protease that plays a central role in vascular homeostasis, and activated recombinant protein C, drotrecogin alfa (activated), has been shown to reduce mortality in patients with severe sepsis. Similar to other serine proteases, functional APC levels are regulated by the serine protease inhibitor family of proteins including α1-antitrypsin and protein C inhibitor. Using APC–substrate modeling, we designed and produced a number of derivatives with the goal of altering the proteolytic specificity of APC such that the variants exhibited resistance to inactivation by protein C inhibitor and α1-antitrypsin yet maintained their primary anticoagulant activity. Substitutions at Leu-194 were of particular interest, because they exhibited 4- to 6-fold reductions in the rate of inactivation in human plasma and substantially increased pharmacokinetic profiles compared with wild-type APC. This was achieved with minimal impairment of the anticoagulant/antithrombotic activity of APC. These data demonstrate the ability to selectively modulate substrate specificity and subsequently affect in vivo performance and suggest therapeutic opportunities for the use of protein C derivatives in disease states with elevated serine protease inhibitor levels.

Role of Protein C in Renal Dysfunction after Polymicrobial Sepsis

Protein C (PC) plays an important role in vascular function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. This study explored the consequences of PC suppression on the kidney in a cecal ligation and puncture model of polymicrobial sepsis. This study shows that a rapid drop in PC after sepsis is strongly associated with an increase in blood urea nitrogen, renal pathology, and expression of known markers of renal injury, including neutrophil gelatinase-associated lipocalin, CXCL1, and CXCL2. The endothelial PC receptor, which is required for the anti-inflammatory and antiapoptotic activity of activated PC (APC), was significantly increased after cecal ligation and puncture as well as in the microvasculature of human kidneys after injury. Treatment of septic animals with APC reduced blood urea nitrogen, renal pathology, and chemokine expression and dramatically reduced the induction of inducible nitric oxide synthase and caspase-3 activation in the kidney. The data demonstrate a clear link between acquired PC deficiency and renal dysfunction in sepsis and suggest a compensatory upregulation of the signaling receptor. Moreover, these data suggest that APC treatment may be effective in reducing inflammatory and apoptotic insult during sepsis-induced acute renal failure.

Activated Protein C Decreases Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand by an EPCR- Independent Mechanism Involving Egr-1/Erk-1/2 Activation

Background—APC is an antithrombotic and antiinflammatory serine protease that plays an important role in vascular function. We report that APC can suppress the proapoptotic mediator TRAIL in human umbilical vein endothelial cells, and we have investigated the signaling mechanism. Methods and Results—APC inhibited endothelial TRAIL expression and secretion and its induction by cell activation. To explore the mechanism, we examined factors associated with TRAIL regulation and demonstrated that APC increased the level of EGR-1, a transcriptional factor known to suppress the TRAIL promoter. APC also induced a significant increase in phosphorylation of ERK-1/2, required to activate EGR-1 expression. Activation of ERK-1/2 was dependent on the protease activated receptor-1 (PAR-1), but independent of the endothelial protein C receptor (EPCR). Using siRNA, we found that the effect of APC on the EGR-1/ERK signaling required for TRAIL inhibition was dependent on the S1P1 receptor and S1P1 kinase. Conclusions—Our data suggest that APC may provide cytoprotective activity by activating the ERK pathway, which upregulates EGR-1 thereby suppressing the expression of TRAIL. Moreover, we provide evidence that APC can induce a cell signaling response through a PAR-1/S1P1-dependent but EPCR-independent mechanism.

Distinct Functions of Activated Protein C Differentially Attenuate Acute Kidney Injury

Administration of activated protein C (APC) protects from renal dysfunction, but the underlying mechanism is unknown. APC exerts both antithrombotic and cytoprotective properties, the latter via modulation of protease-activated receptor-1 (PAR-1) signaling. We generated APC variants to study the relative importance of the two functions of APC in a model of LPS-induced renal microvascular dysfunction. Compared with wild-type APC, the K193E variant exhibited impaired anticoagulant activity but retained the ability to mediate PAR-1-dependent signaling. In contrast, the L8W variant retained anticoagulant activity but lost its ability to modulate PAR-1. By administering wild-type APC or these mutants in a rat model of LPS-induced injury, we found that the PAR-1 agonism, but not the anticoagulant function of APC, reversed LPS-induced systemic hypotension. In contrast, both functions of APC played a role in reversing LPS-induced decreases in renal blood flow and volume, although the effects on PAR-1-dependent signaling were more potent. Regarding potential mechanisms for these findings, APC-mediated PAR-1 agonism suppressed LPS-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. However, the anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics. Both variants reduced active caspase-3 and abrogated LPS-induced renal dysfunction and pathology. We conclude that although PAR-1 agonism is solely responsible for APC-mediated improvement in systemic hemodynamics, both functions of APC play distinct roles in attenuating the response to injury in the kidney.

Negative Regulation of Inducible Nitric-oxide Synthase Expression Mediated through Transforming Growth Factor-β-dependent Modulation of Transcription Factor TCF11

Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-β (TGF-β), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-β1 and by Smad6-short, which enhances TGF-β signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-β inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-β. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-β and Smad6-short could be blocked by staurosporine, and the effect of TGF-β was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.

Modulation of thrombomodulin-dependent activation of human protein C through differential expression of endothelial Smads.

Protein C is a plasma protease that in its active form plays a central role in the regulation of vascular function by modulating thrombosis, inflammation, and apoptosis. A central player in this pathway is the cytokine-regulated receptor thrombomodulin (TM), which functions as a co-factor for the thrombin-dependent generation of activated protein C. We have found that tumor necrosis factor-β (TGF-β)-dependent suppression of TM on endothelial cells is differentially regulated by endothelial Smad6s and Smad7. Overexpression of Smad6s resulted in activation of a TGF-β reporter alone and enhanced TGF-β response. Moreover, Smad6s overexpression suppressed TM and subsequently reduced activated protein C generation. Antisense inhibition of Smad6s expression enhanced the TM-dependent activation of protein C, whereas blocking the inhibitory Smad7 by antisense resulted in reduced TM-dependent activation of protein C. The effect of Smad6s appeared to be due, at least in part, to up-regulation of TGF-β itself. Immunohistochemisty studies in normal versusatherosclerotic vessels showed that TM levels were suppressed in the endothelium over plaque. Consistent with the in vitro data, we found differential expression of Smad6s and Smad7 in normalversus atherosclerotic vessels, with Smad6s expression low in normal vessels but elevated in atherosclerotic vessels. In contrast, the opposite expression pattern was observed for Smad7. Overall, our results suggest that the relative balance of these intracellular Smads modulate the balance of endothelial function with regard to protein C activation.

Development of cloning vehicles from the Streptomyces plasmid pFJ103.

A 20-kb plasmid, pFJ103, was isolated from a strain of Streptomyces granuloruber. A restriction endonuclease map of the plasmid was constructed. A Streptomyces gene that specifies resistance to the antibiotic thiostrepton was subcloned into Escherichia coli plasmid pBR322, inserted into pFJ103 and transformed into Streptomyces ambofaciens protoplasts. Two classes of transformants were obtained. One carries the pFJ104 plasmid consisting of the entire pFJ103 with the 1.8-kb thiostrepton resistance gene insert. The other carries the pFJ105 plasmid consisting of the 2.9-kb replicon segment of pFJ103 with the same thiostrepton resistance insert. A gene for neomycin resistance together with the entire E. coli pBR322 plasmid were cloned into pFJ105. The resulting E. coli-Streptomyces bifunctional vector, pFJ123, transformed both E. coli and Streptomyces. The small size of pFJ105, its ease of isolation, and efficient transformation of Streptomyces protoplasts establishes it, and its derivatives, as useful plasmid cloning vehicles for fundamental and applied studies.

Smad6s Regulates Plasminogen Activator Inhibitor-1 through a Protein Kinase C-β-dependent Up-regulation of Transforming Growth Factor-β

Plasminogen activator inhibitor-1 (PAI-1) is a serpin class protease inhibitor that plays a central role in the regulation of vascular function and tissue remodeling by modulating thrombosis, inflammation, and the extracellular matrix. A central mediator controlling PAI-1 is transforming growth factor-β (TGF-β), which induces its expression and promotes fibrosis. We have found that a unique member of the Smad family of signal transduction molecules, Smad6s, modulates the expression of PAI-1. Overexpression of Smad6s in endothelial cells increases promoter activity and PAI-1 secretion, and an antisense to Smad6s suppresses the induction of PAI-1 by TGF-β. The effect of Smad6s on the PAI-1 promoter appeared to be the result of increase binding of the forkhead winged helix factor FoxD1 to a TGF-β-responsive element. Furthermore, the effect of Smad6s on PAI-1 up-regulation and on FoxD1 binding was found to result from up-regulation of TGF-β and could be inhibited by the blocking TGF-β signaling with Smad7. The ability of Smad6s to regulate the TGF-β promoter and subsequent PAI-1 induction was suppressed by a selective protein kinase C-β (PKC-β) inhibitor. Consistent with the in vitro data, we found that increased Smad6s in diseased vessels correlated with increased TGF-β and PAI-1 levels. Overall, our results demonstrate that the level of Smad6s can alter the level of TGF-β and the subsequent induction of PAI-1 via a FoxD1 transcription site. Furthermore, our data suggest that this process, which is up-regulated in diseased vessels, can be modulated by the inhibition of PKC-β.