Blake J. Cochran

Forty Years Later and the Role of Plasminogen Activator Inhibitor Type 2/SERPINB2 Is Still an Enigma

Plasminogen activator inhibitor (PAI)-2 expression is acutely upregulated in pregnancy, inflammation, infection, and other pathophysiological conditions. Circumstances that prevent PAI-2 upregulation are associated with chronic pathology. Altogether this strongly suggests that PAI-2 is one of the many proteins that maintain homeostasis during damage or stress. However, several functions ranging from a classical serpin to various intracellular roles have been ascribed to PAI-2 and, because none of these have been definitively proven in vivo, to this day its precise role or roles remains an enigma. This review readdresses the evidence supporting a role for PAI-2 in fibrinolysis and proteolysis within extracellular environments and includes a review of the many potential intracellular functions attributed to PAI-2.

The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity

BackgroundPlasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use in vitro of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI-2 ΔCD-loop) for these purposes.ResultsWe show that PAI-2 ΔCD-loop expressed and purified from the pQE9 vector system presents an easier purification target than the previously used pET15b system. Additionally, PAI-2 ΔCD-loop gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions. Importantly, absence of the CD-loop had no impact on the inhibition of both solution phase and cell surface uPA or on the clearance of receptor bound uPA from the cell surface. Furthermore, uPA:PAI-2 ΔCD-loop complexes had similar binding kinetics (KD ~5 nM) with the endocytosis receptor Very Low Density Lipoprotein Receptor (VLDLR) to that previously published for uPA:PAI-2 complexes.ConclusionWe demonstrate that the CD-loop is redundant for the purposes of cellular uPA inhibition and cell surface clearance (endocytosis) and is thus suitable for the development of anti-uPA targeted cancer therapeutics.

Apolipoprotein A-I Increases Insulin Secretion and Production From Pancreatic β-Cells via a G-Protein-cAMP-PKA-FoxO1–Dependent Mechanism

Objective— Therapeutic interventions that increase plasma levels of high-density lipoproteins and apolipoprotein A-I (apoA-I) A-I, the major high-density lipoprotein apolipoprotein, improve glycemic control in people with type 2 diabetes mellitus. High-density lipoproteins and apoA-I also enhance insulin synthesis and secretion in isolated pancreatic islets and clonal &bgr;-cell lines. This study identifies the signaling pathways that mediate these effects. Approach and Results— Incubation with apoA-I increased cAMP accumulation in Ins-1E cells in a concentration-dependent manner. The increase in cAMP levels was inhibited by preincubating the cells with the cell-permeable, transmembrane adenylate cyclase inhibitor, 2′5′ dideoxyadenosine, but not with KH7, which inhibits soluble adenylyl cyclases. Incubation of Ins-1E cells with apoA-I resulted in colocalization of ATP-binding cassette transporter A1 with the G&agr;s subunit of a heterotrimeric G-protein and a G&agr;s subunit-dependent increase in insulin secretion. Incubation of Ins-1E cells with apoA-I also increased protein kinase A phosphorylation and reduced the nuclear localization of forkhead box protein O1 (FoxO1). Preincubation of Ins-1E cells with the protein kinase A–specific inhibitors, H89 and PKI amide, prevented apoA-I from increasing insulin secretion and mediating the nuclear exclusion of FoxO1. Transfection of Ins-1E cells with a mutated FoxO1 that is restricted to the nucleus confirmed the requirement for FoxO1 nuclear exclusion by blocking insulin secretion in apoA-I–treated Ins-1E cells. ApoA-I also increased Irs1, Irs2, Ins1, Ins2, and Pdx1 mRNA levels. Conclusions— ApoA-I increases insulin synthesis and secretion via a heterotrimeric G-protein-cAMP-protein kinase A-FoxO1–dependent mechanism that involves transmembrane adenylyl cyclases and increased transcription of key insulin response and &bgr;-cell survival genes.

Dependence on endocytic receptor binding via a minimal binding motif underlies the differential prognostic profiles of SerpinE1 and SerpinB2 in cancer.

Tumor overexpression of urokinase-type plasminogen activator (uPA) and its specific inhibitor SerpinE1 (plasminogen activator inhibitor type-1) correlates with poor prognosis and increased metastatic potential. Conversely, tumor expression of uPA and another specific inhibitor, SerpinB2 (plasminogen activator inhibitor type-2), are associated with favorable outcome and relapse-free survival. It is not known how overexpression of these uPA inhibitors results in such disparate outcomes. A possible explanation may be related to the presence of a proposed low density lipoprotein receptor (LDLR)-binding motif in SerpinE1 responsible for mitogenic signaling via ERK that is absent in SerpinB2. We now show that complementation of such a LDLR-binding motif in SerpinB2 by mutagenesis of two key residues enabled high affinity binding to very LDLR (VLDLR). Furthermore, the VLDLR-binding SerpinB2 form behaved in a manner indistinguishable from SerpinE1 in terms of enhanced uPA-SerpinB2 complex endocytosis and subsequent ERK phosphorylation and cell proliferation; that is, the introduction of the LDLR-binding motif to SerpinB2 was necessary and sufficient to allow it to acquire characteristics of SerpinE1 associated with malignancy. In conclusion, this study defines the structural elements underlying the distinct interactions of SerpinE1 versus SerpinB2 with endocytic receptors and how differential VLDLR binding impacts on downstream cellular behavior. This has clear relevance to understanding the paradoxical disease outcomes associated with overexpression of these serpins in cancer.

Impact of Perturbed Pancreatic β-Cell Cholesterol Homeostasis on Adipose Tissue and Skeletal Muscle Metabolism

Elevated pancreatic β-cell cholesterol levels impair insulin secretion and reduce plasma insulin levels. This study establishes that low plasma insulin levels have a detrimental effect on two major insulin target tissues: adipose tissue and skeletal muscle. Mice with increased β-cell cholesterol levels were generated by conditional deletion of the ATP-binding cassette transporters, ABCA1 and ABCG1, in β-cells (β-DKO mice). Insulin secretion was impaired in these mice under basal and high-glucose conditions, and glucose disposal was shifted from skeletal muscle to adipose tissue. The β-DKO mice also had increased body fat and adipose tissue macrophage content, elevated plasma interleukin-6 and MCP-1 levels, and decreased skeletal muscle mass. They were not, however, insulin resistant. The adipose tissue expansion and reduced skeletal muscle mass, but not the systemic inflammation or increased adipose tissue macrophage content, were reversed when plasma insulin levels were normalized by insulin supplementation. These studies identify a mechanism by which perturbation of β-cell cholesterol homeostasis and impaired insulin secretion increase adiposity, reduce skeletal muscle mass, and cause systemic inflammation. They further identify β-cell dysfunction as a potential therapeutic target in people at increased risk of developing type 2 diabetes.

Apolipoprotein A-I interactions with insulin secretion and production.

Purpose of review Human population studies have established that an elevated plasma high-density lipoprotein cholesterol (HDL-C) level is associated with a decreased risk of developing cardiovascular disease. In addition to having several potentially cardioprotective functions, HDLs and apolipoprotein (apo)A-I, the main HDL apolipoprotein, also have antidiabetic properties. Interventions that elevate plasma HDL-C and apoA-I levels improve glycemic control in people with type 2 diabetes mellitus by enhancing pancreatic &bgr;-cell function and increasing insulin sensitivity. Recent findings This review is concerned with recent advances in understanding the mechanisms by which HDLs and apoA-I improve pancreatic &bgr;-cell function. Summary HDLs and apoA-I increase insulin synthesis and secretion in pancreatic &bgr; cells. The underlying mechanism of this effect is similar to what has been reported for intestinally derived incretins, such as glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide, which both increase &bgr;-cell insulin secretion under high glucose conditions. This involves the activation of a heterotrimeric G protein G&agr;s subunit on the &bgr;-cell surface that leads to induction of a transmembrane adenylyl cyclase, increased intracellular cyclic adenosine monophosphate and Ca2+ levels, and activation of protein kinase A. Protein kinase A increases insulin synthesis by excluding FoxO1 from the &bgr;-cell nucleus and derepressing transcription of the insulin gene.

The ATP binding cassette transporter, ABCG1, localizes to cortical actin filaments

The ATP-binding cassette sub-family G member 1 (ABCG1) exports cellular cholesterol to high-density lipoproteins (HDL). However, a number of recent studies have suggested ABCG1 is predominantly localised to intracellular membranes. In this study, we found that ABCG1 was organized into two distinct cellular pools: one at the plasma membrane and the other associated with the endoplasmic reticulum (ER). The plasma membrane fraction was organized into filamentous structures that were associated with cortical actin filaments. Inhibition of actin polymerization resulted in complete disruption of ABCG1 filaments. Cholesterol loading of the cells increased the formation of the filamentous ABCG1, the proximity of filamentous ABCG1 to actin filaments and the diffusion rate of membrane associated ABCG1. Our findings suggest that the actin cytoskeleton plays a critical role in the plasma membrane localization of ABCG1.

Inhibition of inflammatory signaling pathways in 3T3-L1 adipocytes by apolipoprotein A-I

Activation of inflammatory signaling pathways links obesity with metabolic disorders. TLR4‐mediated activation of MAPKs and NF‐kB are 2 such pathways implicated in obesity‐induced inflammation. Apolipoprotein A‐I (apoA‐I) exerts anti‐inflammatory effects on adipocytes by effluxing cholesterol from the cells via the ATP binding cassette transporter A1 (ABCA1). It is not known if these effects involve inhibition of inflammatory signaling pathways by apoA‐I. This study asks if apoA‐I inhibits activation of MAPKs and NF‐kB in mouse 3T3‐L1 adipocytes and whether this inhibition is ABCA1 dependent. Incubation of differentiated 3T3‐L1 adipocytes with apoA‐I decreased cell surface expression of TLR4 by 16 ± 2% and synthesis of the TLR4 adaptor protein, myeloid differentiation primary response 88, by 24 ± 4% in an ABCA1‐dependent manner. ApoA‐I also inhibited downstream activation of MAPKs, such as ERK, p38MAPK, and JNK, as well as expression of proinflammatory adipokines in bacterial LPS‐stimulated 3T3‐L1 adipocytes in an ABCA1‐dependent manner. ApoA‐I, by contrast, suppressed nuclear localization of the p65 subunit of NF‐kB by 30 ± 3% in LPS‐stimulated 3T3‐L1 adipocytes in an ABCA1‐independent manner. In conclusion, apoA‐I inhibits TLR4‐mediated inflammatory signaling pathways in adipocytes by preventing MAPK and NF‐kB activation.—Sultana, A., Cochran, B. J., Tabet, F., Patel, M., Cuesta Torres, L., Barter, P. J., Rye, K.‐A. Inhibition of inflammatory signaling pathways in 3T3‐L1 adipocytes by apolipoprotein A‐I. FASEB J. 30, 2324–2335 (2016). www.fasebj.org

The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.

CETP inhibition, statins and diabetes

Type 2 diabetes is a causal risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). While treatment with a statin reduces the risk of having an ASCVD event in all people, including those with type-2 diabetes, statin treatment also increases the likelihood of new onset diabetes when given to those with risk factors for developing diabetes. Treatment with the cholesteryl ester transfer protein (CETP) inhibitor, anacetrapib, reduces the risk of having a coronary event over and above that achieved with a statin. However, unlike statins, anacetrapib decreases the risk of developing diabetes. If the reduced risk of new-onset diabetes is confirmed in another CETP inhibitor outcome trial, there will be a case for considering the use of the combination of a statin plus a CETP inhibitor in high ASCVD-risk people who are also at increased risk of developing diabetes.