THE NEW ROLE OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A CONNECTION OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9, APOLIPOPROTEIN B, and AUTOPHAGY

Plasma low-density lipoprotein (LDL) levels are positively correlated with the incidence of coronary artery disease. In the circulation, the plasma LDL clearance is mainly achieved by the uptake via LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly discovered gene, playing an important role in LDL metabolism. Gain-of-function mutations of PCSK9 lead to hypercholesterolemia and loss-of-function mutations of PCSK9 are associated with decrease of LDL cholesterol. The effects of PCSK9 on cholesterol levels are the consequence of a strong interaction between the catalytic domain of PCSK9 and epidermal growth factor-like repeat A (EGF-A) domain of LDLR on the cell surface of hepatocytes. This PCSK9/LDLR complex enters the cell via endocytosis, where both PCSK9 and LDLR are removed via the lysosome pathway, resulting in decreased levels of LDLR and accumulation of LDL in the plasma. However, whether this is the exclusive function of PCSK9 on LDL metabolism was challenged by us; we observed PCSK9 interacted with apolipoprotein B (apoB) and increased apoB production, irrespective of the LDLR. ApoB is the primary structure protein of LDL particle and it also serves as the ligand for the LDL receptor. There is ample evidence showing that the levels of apoB are a better indicator for heart disease than either total cholesterol or LDL cholesterol levels. We used a second-generation adenoviral vector to overexpress PCSK9 (Ad-PCSK9) in wild-type C57BL/6 and LDLR deficient mice (Ldlr-/- and Ldlr-/-Apobec1-/-). Our study revealed that overexpression of PCSK9 promoted the production and secretion of apoB in the form of very-low density lipoprotein (VLDL), which is the precursor of LDL, in the 3 mouse models studied (C57BL/6J, Ldlr-/-, and Ldlr-/-Apobec1-/-). The increased apoB production in mice was regulated at post-transcriptional levels, since there was no difference in apoB mRNA levels between mice treated with Ad-PCSK9 and control vector Ad-Null. By using pulse-chase experiment on primary hepatocytes, we showed that overexpression of PCSK9 increased the secretion of apoB, independent of LDLR. In the circulation, we showed that PCSK9 was associated with LDL particles. By using 3 different protein–protein interaction assays of co-immunoprecipitation, mammalian two-hybrid system, and in situ proximity ligation assay, we demonstrated a direct protein–protein interaction between PCSK9 and apoB. The impact of this interaction inhibited the physiological removal process of apoB via autophagosome/lysosome pathway in an LDLR-independent fashion, resulting in increased production and secretion of apoB-containing lipoproteins. The significance of this process was shown in the Pcsk9 knockout mice in the background of Ldlr-/-Apobec1-/- mice (triple knockout mice); in the absence of Pcsk9 (triple knockout mice) the levels of cholesterol, triacylglycerol, and apoB decreased significantly in comparison to that of Ldlr-/-Apobec1-/- mice. Taken together, our study demonstrated a direct intracellular interaction of PCSK9 with apoB, resulting in the inhibition of apoB degradation via the autophagosome/lysosome pathway independent of LDLR. This discovery provides a new concept of the importance of PCSK9 and suggests new approaches for the therapeutic intervention of hyperlipidemia.

Stromal derived growth factor alpha (SDF-1α) induces the differentiation of bone marrow progenitor cells (BMCs) into pericytes by regulating platelet derived growth factor B (PDGF-B) transcription

We previously demonstrated that bone marrow cells (BMCs) migrate to TC71 and A4573 Ewing’s sarcoma tumors where they can differentiate into endothelial cells (ECs) and pericytes and, participate in the tumor vascular development. This process of neo-vascularization, known as vasculogenesis, is essential for Ewing’s sarcoma growth with the soluble vascular endothelial growth factor, VEGF165, being the chemotactic factor for BMC migration to the tumor site. Inhibiting VEGF165 in TC71 tumors (TC/siVEGF7-1) inhibited BMC infiltration to the tumor site and tumor growth. Introducing the stromal-derived growth factor (SDF-1α) into the TC/siVEGF7-1 tumors partially restored vasculogenesis with infiltration of BMCs to a perivascular area where they differentiated into pericytes and rescued tumor growth. RNA collected from the SDF-1α-treated TC/siVEGF7-1 tumors also revealed an increase in platelet-derived growth factor B (PDGF-B) mRNA levels. PDGF-B expression is elevated in several cancer types and the role of PDGF-B and its receptor, PDGFR-β, has been extensively described in the process of pericyte maturation. However, the mechanisms by which PDGF-B expression is up-regulated during vascular remodeling and the process by which BMCs differentiate into pericytes during tumor vasculogenesis remain areas of investigation. In this study, we are the first to demonstrate that SDF-1α regulates the expression of PDGF-B via a transcriptional mechanism which involves binding of the ELK-1 transcription factor to the pdgf-b promoter. We are also first to validate the critical role of the SDF-1α/PDGF-B pathway in the differentiation of BMCs into pericytes both in vitro and in vivo. SDF-1α up-regulated PDGF-B expression in both TC/siVEGF7-1 and HEK293 cells. In contrast, down-regulating SDF-1α, down-regulated PDGF-B. We cloned the 2 kb pdgf-b promoter fragment into the pGL3 reporter vector and showed that SDF-1α induced pdgf-b promoter activity. We used chromatin immunoprecipitation (ChIP) and demonstrated that the ELK-1 transcription factor bound to the pdgf-b promoter in response to SDF-1α stimulation in both TC/siVEGF7-1 and HEK293 cells. We collected BMCs from the hind femurs of mice and cultured the cells in medium containing SDF-1α and PDGF-B and found that PDGFR-β+ BMCs differentiated into NG2 and desmin positive pericytes in vitro. In contrast, inhibiting SDF-1α and PDGF-B abolished this differentiation process. In vivo, we injected TC71 or A4573 tumor-bearing mice with the SDF-1α antagonist, AMD3100 and found that inhibiting SDF-1α signaling in the tumor microenvironment decreased the tumor microvessel density, decreased the tumor blood vessel perfusion and, increased tumor cell apoptosis. We then analyzed the effect of AMD3100 on vasculogenesis of Ewing’s sarcoma and found that BMCs migrated to the tumor site where they differentiated into ECs but, they did not form thick perivascular layers of NG2 and desmin positive pericytes. Finally, we stained the AMD3100-treated tumors for PDGF-B and showed that inhibiting SDF-1α signaling also inhibited PDGF-B expression. All together, these findings demonstrated that the SDF-1α/PDGF-B pathway plays a critical role in the formation of BM-derived pericytes during vasculogenesis of Ewing’s sarcoma tumors.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Cotransfer of antibiotic resistance genes and a hylEfm-containing virulence plasmid in Enterococcus faecium.

The hyl(Efm) gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hyl(Efm)-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hyl(Efm) gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hyl(Efm), whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hyl(Efm)-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hyl(Efm) plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

The inhibition of lipid transfer protein by negatively charged liposomes modifies the therapeutic index of amphotericin B

We have shown that liposomal amphotericin B (L-AmpB) decreased renal toxicity and maintains the antifungal activity of amphotericin B (AmpB). We have also observed that L-AmpB is predominantly associated with high density lipoproteins (HDL) as compared to Fungizone (AmpB + deoxycholate). The present experiments were designed to assess the biological relevance of transferring AmpB to HDL. We observed that AmpB was less toxic to kidney cells when associated with HDL, however AmpB toxicity was maintained when associated with LDL. To further understand how HDL-associated AmpB reduces renal cell toxicity the presence of HDL and LDL receptors in this cell line was determined. We observed that these cells expressed high and low affinity LDL receptors, but only low affinity HDL receptors. The reduced renal cell toxicity of HDL-associated AmpB may be due to its lack of interaction with renal cells because of the absence of HDL receptors. Since AmpB interacts with cholesteryl esters whose transfer among lipoproteins is regulated by Lipid transfer Protein (LTP), the role of LTP on the distribution of AmpB to HDL and LDL was next examined. We found that negatively charged liposomes significantly reduced LTP-mediated transfer of CE between HDL and LDL, independent of the presence of AmpB, while Fungizone only significantly inhibited CE transfer at one concentration tested (20$\mu$g/ml). Therefore, we believe that the decreased renal toxicity of L-AmpB is related to its predominant distribution to HDL which is regulated by the inhibition of LTP activity. ^

THE LONG-TERM GROWTH OF NORMAL HUMAN B CELLS

The feasibility of establishment of continuously proliferating growth factor-dependent human B lymphocytes was investigated. Normal B lymphocytes prepared from peripheral venous blood were stimulated with a variety of known polyclonal B cell activators, in the continuous presence of various cytokine preparations. Continuously proliferating growth factor-dependent B cell populations were obtained from cultures activated with either insoluble anti-IgM ((mu)-chain specific), soluble anti-IgM, heat-killed Staphylococcus aureus Cowen I (SAC), or dextran sulphate (DxS), in the continuous presence of exogenously added growth factor preparations containing either IL-1, IL-2 and BCGF, or BCGF alone. Although growth factor-dependent B cell lines were obtained via all three methods of activation, the correlation of mode of activation and growth factor preparation proved to be critical. B cell lines could not be established with anti-(mu) activation in the presence of only BCGF; however, B cell lines were successfully obtained with SAC or DxS activation from those cultures continuously replenished with only BCGF. These cultured B lymphocyte populations were routinely maintained in logarithmic-phase growth in the presence of exogenously added growth factor, and exhibited a population doubling time of approximately 36 hours. They were shown to specifically absorb BCGF, suggesting the presence of membrane receptors for it. Also, these cultured B cells have been utilized for the development of a microassay for the assessment of a M(,r) 12,000-14,000 B cell growth factor activity that is accurate, sensitive, and precise. The pronounced sensitivity of this bioassay beyond that of the conventional peripheral blood B cell assay has aided in the purification to homogeneity of natural product extracellular BCGF (EC-BCGF), and in the determination of the nucleotide sequence for a gene coding for a protein exhibiting BCGF activity. Additionally, these B cell lines specifically absorb, and proliferate in the presence of, an affinity-purified M(,r) 60,000 trypsin-sensitive intracellular protein derived from freshly isolated human T lymphocytes, providing evidence for a putative intracellular precursor of EC-BCGF, or a novel high molecular weight BCGF species. ^

PIM KINASE INHIBITION: SINGLE AGENT AND COMBINATION APPROACHES IN B-CELL LYMPHOMA

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are Ser/Thr/Tyr kinases. They modulate B-cell development but become oncoproteins and promote cancer development once overexpressed. Containing three isoforms, Pim-1, -2 and -3 are known to phosphorylate various substrates that regulate transcription, translation, cell cycle, and survival pathways in both hematological and solid tumors. Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma. Elevated Pim kinase levels are common in MCL, and it negatively correlates with patient outcome. SGI-1776 is a small molecule inhibitor selective for Pim-1/-3. We hypothesize that SGI-1776 treatment in MCL will inhibit Pim kinase function, and inhibition of downstream substrates phosphorylation will disrupt transcriptional, translational, and cell cycle processes while promoting apoptosis. SGI-1776 treatment induced moderate to high levels of apoptosis in four MCL cell lines (JeKo-1, Mino, SP-53 and Granta-519) and peripheral blood mononuclear cells (PBMCs) from MCL patients. Phosphorylation of transcription and translation regulators, c-Myc and 4E-BP1 declined in both model systems. Additionally, levels of short-lived Mcl-1 mRNA and protein also decreased and correlated with decline of global RNA synthesis. Collectively, our investigations highlight Pim kinases as viable drug targets in MCL and emphasize their roles in transcriptional and translational regulation. We further investigated a combination strategy using SGI-1776 with bendamustine, an FDA-approved DNA-damaging alkylating agent for treating non-Hodgkin’s lymphoma. We hypothesized this combination will enhance SGI-1776-induced transcription and translation inhibition, while promoting bendamustine-triggered DNA damage and inducing additive to synergistic cytotoxicity in B-cell lymphoma. Bendamustine alone resulted in moderate levels of apoptosis induction in MCL cell lines (JeKo-1 and Mino), and in MCL and splenic marginal zone lymphoma (a type of B-cell lymphoma) primary cells. An additive effect in cell killing was observed when combined with SGI-1776. Expectedly, SGI-1776 effectively decreased global RNA and protein synthesis levels, while bendamustine significantly inhibited DNA synthesis and generated DNA damage response. In combination, intensified inhibitory effects in DNA, RNA and protein syntheses were observed. Together, these data suggested feasibility of using Pim kinase inhibitor in combination with chemotherapeutic agents such as bendamustine in B-cell lymphoma, and provided foundation of their mechanism of actions in lymphoma cells.

CHARACTERIZATION OF THE ROLE OF CARMA3 IN ENDOPLASMIC RETICULUM STRESS-INDUCED NF-κB ACTIVATION

Endoplasmic reticulum (ER) stress-induced inflammation plays an important role in the progression of many diseases, such as type II diabetes, insulin resistance, cancers, and so on. NF-κB is believed to be a central regulator of ER stress-induced inflammation. However, studies on how ER stress induces NF-κB activation are limited and, in some cases, controversial. In the present study, we utilized two commonly used ER stress inducers, thapsigargin and tunicamycin, to study the mechanism. We found that two caspase-recruitment domain (CARD)-containing proteins, CARMA3 and BCL10, play a crucial roles on ER stress-induced NF-κB activation by regulating IκBα kinase activity. Consistently, we observed that a physiological ER stress inducer, hypoxia, could activate NF-κB in a CARMA3-dependent manner. Additionally, we showed that the activation of the UPR signaling pathways were intact in both CARMA3- and BCL10-deficient cells under ER stress. Together, this study provides insight into the mechanism of how ER stress induces NF-κB activation. It allows us to better understand ER stress-induced inflammation and develop the corresponding therapeutic interference to treat diseases