Jong-Sup Bae

Structural Coupling of Smad and Runx2 for Execution of the BMP2 Osteogenic Signal

Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-β (TGFβ) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426-428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGFβ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity.

Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells

Ever-increasing evidence in the literature suggests that the antiinflammatory and cytoprotective properties of activated protein C (APC) are mediated through its endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells. However, recent results monitoring the cleavage rate of PAR-1 on human umbilical vein endothelial cells, transfected with an alkaline phosphatase–PAR-1 fusion reporter construct, have indicated that the catalytic activity of thrombin toward PAR-1 is several orders of magnitude higher than that of APC. Because thrombin is required for generation of APC, and because it also functions in the proinflammatory pathways through the activation of PAR-1, it has been difficult to understand how APC can elicit protective cellular responses through the activation of PAR-1 when thrombin is present. In this study we provide a plausible answer to this question by demonstrating that the critical receptors required for both protein C activation (thrombomodulin and EPCR) and APC cellular signaling (EPCR and PAR-1) pathways are colocalized in the membrane lipid rafts in endothelial cells. We further show that the APC cleavage of PAR-1 on cells transfected with a PAR-1 cleavage reporter construct is not sensitive to the cofactor function of EPCR. Thus, the colocalization of EPCR and PAR-1 in lipid rafts is a key requirement for the cellular signaling activity of APC. Thrombomodulin colocalization with these receptors on the same membrane microdomain can also recruit thrombin to activate the EPCR-bound protein C, thereby eliciting PAR-1 signaling events that are involved in the APC protective pathways.

Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand

We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by its natural ligand activated protein C (APC)/protein C switches the protease activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin from a disruptive to a protective effect in cultured human umbilical vein endothelial cells. Given the phenotypic differences between endothelial cells in venular and arterial beds, in this study we evaluated the signaling function of thrombin in human pulmonary artery endothelial cells (HPAECs) before and after treating them with PC-S195A which lacks catalytic activity but exhibits a normal affinity for EPCR. As expected, both thrombin and thrombin receptor agonist peptide (TRAP) enhanced the permeability barrier of HPAECs; however, both PAR-1 agonists exhibited a potent barrier protective effect when the cells were treated with PC-S195A prior to stimulation by the agonists. Interestingly, similar to APC, thrombin exhibited a potent cytoprotective activity in the LPS-induced permeability and TNF-alpha-induced apoptosis and adhesion assays in the PC-S195A treated HPAECs. Treatment of HPAECs with the cholesterol depleting molecule methyl-beta-cyclodextrin eliminated the protective effect of both APC and thrombin. These results suggest that the occupancy of EPCR by its natural ligand recruits PAR-1 to a protective signaling pathway within lipid rafts of HPAECs. Based on these results we conclude that the activation of PAR-1 by thrombin would initiate a protective response in intact arterial vascular cells expressing EPCR. These findings may have important ramifications for understanding the mechanism of the participation of the vascular PAR-1 in pathophysiology of the inflammatory disorders.

Engineering a Disulfide Bond to Stabilize the Calcium-binding Loop of Activated Protein C Eliminates Its Anticoagulant but Not Its Protective Signaling Properties

In addition to an anticoagulant activity, activated protein C (APC) also exhibits anti-inflammatory and cytoprotective properties. These properties may contribute to the beneficial effect of APC in treating severe sepsis patients. A higher incidence of bleeding because of its anticoagulant function has been found to be a major drawback of APC as an effective anti-inflammatory drug. In this study, we have prepared a protein C variant in which an engineered disulfide bond between two β-sheets stabilized the functionally critical Ca2+-binding 70-80 loop of the molecule. The 70-80 loop of this mutant no longer bound Ca2+, and the activation of the mutant by thrombin was enhanced 60-80-fold independently of thrombomodulin. The anticoagulant activity of the activated protein C mutant was nearly eliminated as determined by a plasma-based clotting assay. However, the endothelial protein C receptor- and protease-activated receptor-1-dependent protective signaling properties of the mutant were minimally altered as determined by staurosporine-induced endothelial cell apoptosis, thrombin-induced endothelial cell permeability, and tumor necrosis-α-mediated neutrophil adhesion and migration assays. These results suggest that the mutant lost its ability to interact with the procoagulant cofactors but not with the protective signaling molecules; thus this mutant provides an important tool for in vivo studies to examine the role of anticoagulant versus anti-inflammatory function of activated protein C.

Reconstitution of Runx2/Cbfa1‐null cells identifies a requirement for BMP2 signaling through a Runx2 functional domain during osteoblast differentiation

The Runx2/Cbfa1 transcription factor is a scaffolding protein that promotes osteoblast differentiation; however, the specific Runx2‐functional domains required for induction of the osteogenic lineage remain to be identified. We approached this question using a TERT‐immortalized cell line derived from calvaria of Runx2‐null mice by reconstituting the osteogenic activity with wild‐type and deletion mutants of Runx2. The presence or absence of osteogenic media (β‐glycerol phosphate and ascorbic acid) and/or with BMP2 did not stimulate osteoblastic gene expression in the Runx2‐null cells. However, cells infected with wild‐type Runx2 adenovirus showed a robust temporal increase in the expression of osteoblast marker genes and were competent to respond to BMP2. Early markers (i.e., collagen type‐1, alkaline phosphatase) were induced (four‐ to eightfold) at Days 4 and 8 of culture. Genes representing mature osteoblasts (e.g., Runx2, osteopontin, bone sialoprotein, osteocalcin) were temporally expressed and induced from 18‐ to 36‐fold at Days 8 and 12. Interestingly, TGFβ and Vitamin D‐mediated transcription of osteoblast genes (except for osteopontin) required the presence of Runx2. Runx2 lacking the C‐terminal 96 amino acids (Runx2 Δ432) showed a pattern of gene expression similar to wild‐type protein, demonstrating the Groucho interaction and part of the activation domain are dispensable for Runx2 osteogenic activity. Upon further deletion of the Runx2 C‐terminus containing the nuclear matrix targeting signal and Smad‐interacting domain (Δ391), we find none of the osteoblast markers are expressed. Therefore, the Runx2 391‐432 domain is essential for execution of the BMP2 osteogenic signal. J. Cell. Biochem. 100: 434–449, 2007.

Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells

Summary.  Background: The endothelial protein C receptor (EPCR)‐dependent cleavage of protease activated receptor 1 (PAR‐1) by either activated protein C (APC) or thrombin in lipid rafts initiates protective signaling responses in endothelial cells. Objectives: To investigate the mechanism by which APC and thrombin interact with and cleave PAR‐1 in lipid rafts. Methods: We constructed two types of PAR‐1 cleavage reporter constructs in which a secreted alkaline phosphatase (ALP) was fused to the extracellular domain of PAR‐1. The first construct has a transmembrane domain capable of uniformly anchoring the fusion protein to the membrane surface, while the second construct has the recognition sequence for targeting the fusion protein to lipid rafts/caveolae in transfected cells. Results: Both APC and the Gla‐domainless (GD)‐APC cleaved the PAR‐1 exodomain with similar efficiency in HUVECs transfected with the first construct. Unlike APC, GD‐APC did not cleave PAR‐1 in cells transfected with the second construct; however, prior treatment of cells with S195A mutants of either protein C or thrombin led to the GD‐APC cleavage of PAR‐1 with a comparable or higher catalytic efficiency. The same results were obtained if the cellular signaling properties of APC and GD‐APC were monitored in the TNF‐α‐induced endothelial cell apoptosis and permeability assays. Conclusions: The lipid raft localization renders the scissile bond of the PAR‐1 exodomain unavailable for interaction with coagulation proteases. The binding of either the Gla‐domain of protein C to EPCR or exosite‐1 of thrombin to the C‐terminal hirudin‐like sequence of PAR‐1 changes the membrane localization and/or the conformation of the PAR‐1 exodomain to facilitate its recognition and subsequent cleavage by these proteases.

Specific Residues of RUNX2 Are Obligatory for Formation of BMP2-Induced RUNX2-SMAD Complex to Promote Osteoblast Differentiation

BMP2 signaling and RUNX2 regulatory pathways converge for transcriptional control of bone formation in vivo. SMAD proteins are recruited to RUNX2 regulatory complexes via an overlapping nuclear matrix targeting signal/Smad interacting domain sequence (391–432) in Runx2. To establish the contribution of RUNX2-SMAD interaction to osteoblastogenesis, we characterized a number of point mutants. Only a triple mutation of amino acids 426–428 (HTY-AAA) results in loss of RUNX2 interactions with either BMP2- or TGF-β- responsive SMADs and fails to integrate the BMP2/TGF-β signal on target gene promoters. In a Runx2 null cell reconstitution assay, the HTY mutant did not activate the program of osteoblast differentiation (alkaline phosphatase, collagen type 1, osteopontin, bone sialoprotein and osteocalcin) in response to BMP2 signaling. Thus, subnuclear targeting function and formation of a RUNX2-SMAD osteogenic complex are functionally inseparable. Taken together, these studies provide direct evidence that RUNX2 is essential for execution and completion of BMP2 signaling for osteoblast differentiation.

Identification of a specific exosite on activated protein C for interaction with protease-activated receptor 1.

Activated protein C (APC) is a vitamin K-dependent plasma serine protease which down-regulates the clotting cascade by inactivating procoagulant factors Va and VIIIa by limited proteolysis. In addition to its anticoagulant effect, APC also exhibits cytoprotective and antiinflammatory activity through the endothelial protein C receptor-dependent cleavage of protease activated receptor 1 (PAR-1) on endothelial cells. Recent mutagenesis data have indicated that the basic residues of two surface loops including those on 39 and the Ca2+-binding 70–80 loops constitute interactive sites for both factors Va and VIIIa, thereby mediating the interaction of APC specifically with these procoagulant cofactors. The basic residues of both loops have been discovered to be dispensable for the interaction of APC with PAR-1. It is not known if a similar exosite-dependent interaction contributes to the specificity of APC recognition of PAR-1 on endothelial cells. In this study, we have identified two acidic residues on helix-162 (Glu-167 and Glu-170) on the protease domain of APC which are required for the protease interaction with PAR-1, but not for its interaction with the procoagulant cofactors. Thus, the substitution of either Glu-167 or Glu-170 with Ala eliminated the cytoprotective signaling properties of APC without affecting its anticoagulant activity. These mutants provide useful tools for initiating in vivo studies to understand the extent to which the anticoagulant versus antiinflammatory activity of APC contributes to its beneficial effect in treating severe sepsis.

Thrombin inhibits nuclear factor κB and RhoA pathways in cytokine-stimulated vascular endothelial cells when EPCR is occupied by protein C

The occupancy of endothelial protein C receptor (EPCR) by protein C switches the protease activated receptor 1 (PAR-1)-dependent signalling specificity of thrombin from a permeability enhancing to a barrier protective response in vascular endothelial cells. In this study, the modulatory effects of thrombin and thrombin receptor agonist peptides (TRAP) on tumour necrosis factor (TNF)-alpha-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A were evaluated by monitoring the expression of cell surface adhesion molecules (VCAM-1, ICAM-1 and E-selectin), adhesion of freshly isolated neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factor-kappaB (NF-kappaB) pathway. The analysis of results indicate that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither PAR-1 agonist influenced the proinflammatory effects of TNF-alpha in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin, thus leading to thrombin inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNF-alpha-activated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kappaB pathways in response to TNF-alpha in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is ligated by protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses, thus leading to activation of Rac1 and inhibition of RhoA and NF-kappaB signalling cascades in vascular endothelial cells.

Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite

Ixolaris is a two‐Kunitz tick salivary gland tissue factor pathway inhibitor (TFPI). In contrast to human TFPI, Ixolaris specifically binds to factor Xa (FXa) heparin‐binding exosite (HBE). In addition, Ixolaris interacts with zymogen FX. In the present work we characterized the interaction of Ixolaris with human FX quantitatively, and identified a precursor state of the heparin‐binding exosite (proexosite, HBPE) as the Ixolaris‐binding site on the zymogen. Gel‐filtration chromatography demonstrated 1:1 complex formation between fluorescein‐labeled Ixolaris and FX. Isothermal titration calorimetry confirmed that the binding of Ixolaris to FX occurs at stoichiometric concentrations in a reaction which is characteristically exothermic, with a favorable enthalpy (ΔH) of −10.78 kcal/mol. ELISA and plasmon resonance experiments also indicate that Ixolaris binds to plasma FX and FXa, or to recombinant Gla domain‐containing FX/FXa with comparable affinities (∼1 nM). Using a series of mutants on the HBPE, we identified the most important amino acids involved in zymogen/Ixolaris interaction—Arg‐93 >>> Arg‐165 ≥ Lys‐169 > Lys‐236 > Arg‐125—which was identical to that observed for FXa/Ixolaris interaction. Remarkably, Ixolaris strongly inhibited FX activation by factor IXa in the presence but not in the absence of factor VIIIa, suggesting a specific interference in the cofactor activity. Further, solid phase assays demonstrated that Ixolaris inhibits FX interaction with immobilized FVIIIa. Altogether, Ixolaris is the first inhibitor characterized to date that specifically binds to FX HBPE. Ixolaris may be a useful tool to study the physiological role of the FX HBPE and to evaluate this domain as a target for anticoagulant drugs.