Jiaxuan Chen

Protein-disulfide Isomerase-associated 3 (Pdia3) Mediates the Membrane Response to 1,25-Dihydroxyvitamin D3 in Osteoblasts

Protein-disulfide isomerase-associated 3 (Pdia3) is a multifunctional protein hypothesized to be a membrane receptor for 1,25(OH)2D3. In intestinal epithelium and chondrocytes, 1,25(OH)2D3 stimulates rapid membrane responses that are different from genomic effects via the vitamin D receptor (VDR). In this study, we show that 1,25(OH)2D3 stimulates phospholipase A2 (PLA2)-dependent rapid release of prostaglandin E2 (PGE2), activation of protein kinase C (PKC), and regulation of bone-related gene transcription and mineralization in osteoblast-like MC3T3-E1 cells (WT) via a mechanism involving Pdia3. Pdia3 was present in caveolae based on co-localization with lipid rafts and caveolin-1. In Pdia3-silenced (Sh-Pdia3) cells, 1,25(OH)2D3 failed to stimulate PKC and PGE2 responses; in Pdia3-overexpressing cells (Ov-Pdia3), responses to 1,25(OH)2D3 were augmented. Downstream mediators of Pdia3, PLA2-activating protein (PLAA) and arachidonic acid, stimulated similar PKC activation in wild-type, Sh-Pdia3, and Ov-Pdia3 cells supporting the hypothesis that Pdia3 mediates the membrane action of 1,25(OH)2D3. Treatment of MC3T3-E1 cells with 1,25(OH)2D3 for 9 min stimulated rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and increased expression of alkaline phosphatase, MMP-13, and osteopontin but decreased expression of osteocalcin, osteoprotegerin (mRNA and protein), and smad2. These effects were attenuated in Sh-Pdia3 cells. Sh-Pdia3 cells produced higher numbers of von Kossa-positive nodules and alizarin red-positive nodules compared with WT cells with or without 1,25(OH)2D3 treatment whereas Ov-Pdia3 did not show any mineralization. Our data suggest Pdia3 is an important initiator of 1,25(OH)2D3-stimulated membrane signaling pathways, which have both genomic and non genomic effects during osteoblast maturation.

Disruption of Pdia3 gene results in bone abnormality and affects 1α,25-dihydroxy-vitamin D3-induced rapid activation of PKC☆

1,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3] is a critical regulator of bone development. Protein disulfide isomerase A3 (Pdia3) is a multifunctional protein that has been associated with rapid membrane-initiated signalling by 1alpha,25(OH)2D3 in several cell types. To identify the physiological roles of Pdia3 in skeletal development, we generated Pdia3-deficient mice. No homozygous mice were observed at birth, indicating that the targeted disruption of the Pdia3 gene resulted in embryonic lethality. Pdia3 deficiency also resulted in skeletal manifestations as revealed by muCT analysis of femurs from 15-week-old heterozygous mice. The Pdia3+/- mice had increased metaphyseal bone volume and trabeculae compared to Pdia3+/+ mice. In contrast, the area and thickness of cortical bone at the femoral mid-diaphysis of Pdia3+/+ mice significantly exceeded that of Pdia3+/- mice. In vitro studies in osteoblast-like MC3T3-E1 cells showed that silencing of Pdia3 abolished 1alpha,25(OH)2D3-induced rapid activation of protein kinase C (PKC) while overexpression of Pdia3 resulted in augmentation of PKC activity by 1alpha,25(OH)2D3. Taken together, these data indicated that Pdia3 plays a crucial role in 1alpha,25(OH)2D3-regulated bone formation and the Pdia3-PKC signalling pathway might be involved in this process.

Plasma membrane Pdia3 and VDR interact to elicit rapid responses to 1α,25(OH)2D3

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) regulates osteoblasts through genomic and rapid membrane-mediated responses. Here we examined the interaction of protein disulfide isomerase family A, member 3 (Pdia3) and the traditional vitamin D receptor (VDR) in plasma membrane-associated responses to 1α,25(OH)2D3. We found that Pdia3 co-localized with VDR and the caveolae scaffolding protein, caveolin-1 on the surface of MC3T3-E1 osteoblasts. Immunoprecipitation showed that both Pdia3 and VDR interacted with caveolin-1. Pdia3 further interacted with phospholipase A2 activating protein (PLAA), whereas VDR interacted with c-Src. 1α,25(OH)2D3 changed the interactions and transport of the two receptors and rapidly activated phospholipase A2 (PLA2) and c-Src. Silencing either receptor or caveolin-1 inhibited both PLA2 and c-Src, indicating that the two receptors function interdependently. These two receptor dependent rapid responses to 1α,25(OH)2D3 regulated gene expression, proliferation and apoptosis of MC3T3-E1 cells. These data demonstrate the importance of both receptors and caveolin-1 in mediating membrane responses to 1α,25(OH)2D3 and subsequently regulating osteoblast biology.

Mechanism of Pdia3-dependent 1α,25-dihydroxy vitamin D3 signaling in musculoskeletal cells

1α,25-Dihydroxy vitamin D3 [1,25(OH)2D3] acts on cells through traditional steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-associated signaling pathways. Two receptors have been implicated in rapid signaling by 1,25(OH)2D3, the classical nuclear vitamin D receptor (VDR) and the more recently identified protein disulfide isomerase, family A, member 3 (Pdia3). Our lab along with other groups has established various tools to investigate the role of these two receptors, including gene knock-out, conditional knock-out, silencing, and over-expression in various model systems (growth plate chondrocytes, osteoblastic cells, chick intestinal epithelial cells, mouse embryoid bodies, extracellular matrix vesicles and isolated cell membranes). The data demonstrate the requirement for Pdia3 in 1,25(OH)2D3 induced phospholipase A2 (PLA2) and protein kinase C (PKC) activation and downstream responses. Pdia3+/- heterozygote mice also exhibit both cartilage and bone defects. VDR is present on the plasma membrane and one VDR-/- mouse strain lacks transcaltachia, although 1,25(OH)2D3 induced PKC activation and transcaltachia are not affected in another VDR-/- mouse strain. In the context of osteoblast differentiation, both receptors are expressed during osteogenic commitment of embryoid bodies and silencing of each causes a more mature osteoblast phenotype in MC3T3-E1 pre-osteoblasts. Pdia3 exists in caveolae, where it interacts with PLA2 activating protein (PLAA) and caveolin-1 to initiate rapid signaling via PLA2, phospholipase C (PLC), PKC, and ultimately the ERK1/2 family of mitogen activated protein kinases (MAPK). Using the growth plate chondrocyte and matrix vesicle models, we have demonstrated that Pdia3-dependent signaling in response to 1,25(OH)2D3 regulates growth plate physiology.

C-terminal domain of SARS-CoV main protease can form a 3D domain-swapped dimer

SARS coronavirus main protease (Mpro) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti‐SARS drug development. We have reported that both the Mpro C‐terminal domain alone (Mpro‐C) and the N‐finger deletion mutant of Mpro (Mpro‐Δ7) exist as a stable dimer and a stable monomer (Zhong et al., J Virol 2008; 82:4227‐4234). Here, we report structures of both Mpro‐C monomer and dimer. The structure of the Mpro‐C monomer is almost identical to that of the C‐terminal domain in the crystal structure of Mpro. Interestingly, the Mpro‐C dimer structure is characterized by 3D domain‐swapping, in which the first helices of the two protomers are interchanged and each is enwrapped by four other helices from the other protomer. Each folding subunit of the Mpro‐C domain‐swapped dimer still has the same general fold as that of the Mpro‐C monomer. This special dimerization elucidates the structural basis for the observation that there is no exchange between monomeric and dimeric forms of Mpro‐C and Mpro‐Δ7.

Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors

1α,25‐Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein‐2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three‐dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild‐type (WT), Pdia3‐silenced (Sh‐Pdia3) and VDR‐silenced (Sh‐VDR) pre‐osteoblastic MC3T3‐E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε‐caprolactone)–collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro‐computed tomography (μCT) showed that the mineralized volume of untreated Sh‐Pdia3 and Sh‐VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh‐VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3. Copyright

Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses. The main protease (Mpro) of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing. Mpro is initially produced as part of viral polyproteins and it is matured by autocleavage. Here, we report that, with the addition of an N-terminal extension peptide, Mpro can form a domain-swapped dimer. After complete removal of the extension peptide from the dimer, the mature Mpro self-assembles into a novel super-active octamer (AO-Mpro). The crystal structure of AO-Mpro adopts a novel fold with four domain-swapped dimers packing into four active units with nearly identical conformation to that of the previously reported Mpro active dimer, and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains. Compared with the previously well characterized form of Mpro, in equilibrium between inactive monomer and active dimer, the stable AO-Mpro exhibits much higher proteolytic activity at low concentration. As all eight active sites are bound with inhibitors, the polyvalent nature of the interaction between AO-Mpro and its polyprotein substrates with multiple cleavage sites, would make AO-Mpro functionally much more superior than the Mpro active dimer for polyprotein processing. Thus, during the initial period of SARS-CoV infection, this novel active form AOMpro should play a major role in cleaving polyproteins as the protein level is extremely low. The discovery of AOMpro provides new insights about the functional mechanism of Mpro and its maturation process.

Without Its N-Finger, the Main Protease of Severe Acute Respiratory Syndrome Coronavirus Can Form a Novel Dimer through Its C-Terminal Domain

ABSTRACT The main protease (Mpro) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. It was found that SARS-CoV Mpro exists in solution as an equilibrium of both monomeric and dimeric forms, and the dimeric form is the enzymatically active form. However, the mechanism of SARS-CoV Mpro dimerization, especially the roles of its N-terminal seven residues (N-finger) and its unique C-terminal domain in the dimerization, remain unclear. Here we report that the SARS-CoV Mpro C-terminal domain alone (residues 187 to 306; Mpro-C) is produced in Escherichia coli in both monomeric and dimeric forms, and no exchange could be observed between them at room temperature. The Mpro-C dimer has a novel dimerization interface. Meanwhile, the N-finger deletion mutant of SARS-CoV Mpro also exists as both a stable monomer and a stable dimer, and the dimer is formed through the same C-terminal-domain interaction as that in the Mpro-C dimer. However, no C-terminal domain-mediated dimerization form can be detected for wild-type SARS-CoV Mpro. Our study results help to clarify previously published controversial claims about the role of the N-finger in SARS-CoV Mpro dimerization. Apparently, without the N-finger, SARS-CoV Mpro can no longer retain the active dimer structure; instead, it can form a new type of dimer which is inactive. Therefore, the N-finger of SARS-CoV Mpro is not only critical for its dimerization but also essential for the enzyme to form the enzymatically active dimer.

Syndecan-1 Modulates the Motility and Resolution Responses of Macrophages

Objective—Syndecan-1 (Sdc-1) is a member of a family of cell surface proteoglycans, which has been reported to participate in the regulation of events relevant to tissue repair and chronic injury responses, including cell–substrate interactions, matrix remodeling, and cell migration. In this study, we report the functional significance of Sdc-1 in polarized macrophage populations and its role in adhesion and motility events relevant to resolution of the inflammatory program. Approach and Results—Macrophage Sdc-1 expression is associated with differentiated M2 macrophages with high intrinsic motility, and Sdc-1 deficiency is characterized by impaired migration and enhanced adhesion. Leukocyte infiltration and emigration were examined in a thioglycollate-induced model of peritonitis in Sdc-1+/+ and Sdc-1−/− mice. Although the infiltration of inflammatory cells was similar in both cohorts, a significant delay in the lymphatic clearance of Sdc-1−/− macrophages was observed. Moreover, we observed enhanced inflammation and greater burden of atherosclerotic plaques in ApoE−/−Sdc-1−/− mice maintained on a Western diet. Conclusions—These results demonstrate that defective motility in Sdc-1−/− macrophages promotes a persistent inflammatory state with relevance to the pathogenesis of atherosclerosis.

Efferocytosis as a regulator of macrophage chemokine receptor expression and polarization

Efferocytosis has been suggested to promote macrophage resolution programs that are dependent on motility and emigration, however, few studies have addressed directed migration in resolving macrophages. In this report, we hypothesized that efferocytosis would induce differential chemokine receptor expression. Polarized macrophage populations, including macrophages actively engaged in efferocytosis, were characterized by PCR array and traditional transwell motility assays. We identified specific up‐regulation of chemokine receptor CXCR4 on both mouse and human macrophages and characterized in vivo expression of CXCR4 in a resolving model of murine peritonitis. Using adoptive transfer and AMD3100 blocking, we confirmed a role for CXCR4 in macrophage egress to draining lymphatics. Collectively these data provide an important mechanistic link between efferocytosis and macrophage emigration.