Inhibitor of differentiation-3 mediates high fat diet-induced visceral fat expansion.

OBJECTIVE: Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. METHODS AND RESULTS: Id3(-/-) mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3(-/-) mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. CONCLUSIONS: Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.

Targeting of Acyl-CoA synthetase 5 decreases jejunal fatty acid activation with no effect on dietary long-chain fatty acid absorption.

BACKGROUND: The absorption of dietary long chain fatty acids (LCFA) largely occurs in the jejunum. LCFA are activated via conjugation with Coenzyme A (CoA), a reaction catalyzed by Acyl-CoA synthetases (ACS). Acyl-CoA sythesis is critical for dietary LCFA absorption; yet, the jejunal ACS enzymes that catalyze the reaction are largely unknown. FINDINGS: High throughput mRNA sequencing of the mouse jejunum revealed that the expression of acyl-CoA synthetase 5 (Acsl5) and fatty-acid transport protein 4 (Fatp4) largely exceeded all other annotated ACS genes that activate LCFA. Interestingly, Acsl5 knockout (KO) mice displayed a decrease of 60% in jejunal total long chain acyl-CoA synthesis rate. Nevertheless, and despite of this decrease, dietary LCFA absorption and body-weight gain in response to high fat diet remained unaffected. CONCLUSION: Acsl5 is a major activator of dietary LCFA, yet in Acsl5 KO mice residual ACS activity is sufficient for maintaining a normal LCFA absorption. Our findings provide further evidence for a robust small intestine LCFA absorption capacity.

Tyrosine phosphorylation as a conformational switch: a case study of integrin ß3 cytoplasmic tail.

Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, α(IIb)ß(3) integrin. We have analyzed phosphorylated (Tyr(747) and Tyr(759)) ß(3) integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr(747) phosphorylation prevents ß(3)CT from binding to its heterodimer partner α(IIb)CT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of ß(3)CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation.

Id3 is a novel atheroprotective factor containing a functionally significant single-nucleotide polymorphism associated with intima-media thickness in humans.

RATIONALE: The gene encoding the helix-loop-helix transcription factor Id3 (inhibitor of differentiation-3) is located within atherosclerosis susceptibility loci of both mice and humans, yet its influence on atherosclerosis is not known. OBJECTIVE: The present study sought to determine whether polymorphisms in the ID3 gene were associated with indices of atherosclerosis in humans and if loss of Id3 function modulated atherogenesis in mice. METHODS AND RESULTS: Six tagging single-nucleotide polymorphisms (SNPs) (tagSNPs) in the human ID3 gene were assessed in participants of the Diabetes Heart Study. One tagSNP, rs11574, was independently associated with carotid intima-media thickness (IMT). The human ID3 variant at rs11574 results in an alanine to threonine substitution in the C terminus. To determine the effect of this polymorphism on the basic function of Id3, site-directed mutagenesis of the human ID3 gene at rs11574 was performed. Results demonstrated a significant reduction in coimmunoprecipitation of the known E-protein partner, E12, with Id3 when it contains the sequence encoded by the risk allele (Id3105T). Further, Id3105T had an attenuated ability to modulate E12-mediated transcriptional activation compared to Id3 containing the ancestral allele (Id3105A). Microarray analysis of vascular smooth muscle cells from WT and Id3(-/-) mice revealed significant modulation of multiple gene pathways implicated in atherogenesis. Moreover, Id3(-/-)ApoE(-/-) mice developed significantly more atherosclerosis in response to 32 weeks of Chow or Western diet feeding than Id3(+/+)ApoE(-/-) mice. CONCLUSIONS: Taken together, results provide novel evidence that Id3 is an atheroprotective factor and link a common SNP in the human ID3 gene to loss of Id3 function and increased IMT.

Increased 12/15-lipoxygenase enhances cell growth, fibronectin deposition, and neointimal formation in response to carotid injury.

OBJECTIVE: To determine whether increased 12/15-lipoxygenase (12/15LO) expression in vivo enhances neointimal formation in response to injury. METHODS AND RESULTS: 12/15LO expression in the vessel wall is increased in animal models of metabolic syndrome and diabetes mellitus. Increased expression of 12/15LO enhances cultured vascular smooth muscle cell (VSMC) proliferation, an effect mediated by the helix-loop-helix factor inhibitor of differentiation 3 (Id3). Carotid endothelial denudation was performed on apolipoprotein (Apo) E(-/-), ApoE(-/-)/12/15LO(-/-), C57BL/6, and 12/15LO-overexpressing transgenic mice. ApoE(-/-)/12/15LO(-/-) mice had attenuated and 12/15LO-overexpressing transgenic mice had enhanced neointimal formation compared with control mice. 12/15LO-overexpressing transgenic mice had greater postinjury carotid Id3 and Ki-67 expression, cell number, and fibronectin deposition compared with C57BL/6 mice. Loss of 12/15LO attenuated proliferation of cultured ApoE(-/-) VSMCs, whereas 12/15LO overexpression induced VSMC proliferation. Loss of Id3 enhanced immunoglobulin trascription factor (ITF)-2b binding to and activation of the p21(cip1) promoter and abrogated 12/15LO-induced VSMC proliferation. CONCLUSIONS: To our knowledge, these data are the first demonstration that increased expression of 12/15LO in the vessel wall enhances Id3-dependent cell proliferation, fibronectin deposition, and neointimal formation in response to injury. Results identify p21(cip1) as a potential target of the 12/15LO-Id3 pathway and suggest that modulation of this pathway may have therapeutic implications for targeting the increased risk of restenosis in patients with diabetes.

Zizimin1, a novel Cdc42 activator, reveals a new GEF domain for Rho proteins.

The Rho family GTPases Rac, Rho and Cdc42 are critical in regulating the actin-based cytoskeleton, cell migration, growth, survival and gene expression. These GTPases are activated by guanine nucleotide-exchange factors (GEFs). A biochemical search for Cdc42 activators led to the cloning of zizimin1, a new protein whose overexpression induces Cdc42 activation. Sequence comparison combined with mutational analysis identified a new domain, which we named CZH2, that mediates direct interaction with Cdc42. CZH2-containing proteins constitute a new superfamily that includes the so-called 'CDM' proteins that bind to and activate Rac. Together, the results suggest that CZH2 is a new GEF domain for the Rho family of proteins.

Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI.

The proper function of Rho GTPases requires precise spatial and temporal regulation of effector interactions. Integrin-mediated cell adhesion modulates the interaction of GTP-Rac with its effectors by controlling GTP-Rac membrane targeting. Here, we show that the translocation of GTP-Rac to membranes is independent of effector interactions, but instead requires the polybasic sequence near the carboxyl terminus. Cdc42 also requires integrin-mediated adhesion for translocation to membranes. A recently developed fluorescence resonance energy transfer (FRET)-based assay yields the surprising result that, despite its uniform distribution, the interaction of activated V12-Rac with a soluble, cytoplasmic effector domain is enhanced at specific regions near cell edges and is induced locally by integrin stimulation. This enhancement requires Rac membrane targeting. We show that Rho-GDI, which associates with cytoplasmic GTP-Rac, blocks effector binding. Release of Rho-GDI after membrane translocation allows Rac to bind to effectors. Thus, Rho-GDI confers spatially restricted regulation of Rac-effector interactions.

The helix-loop-helix factors Id3 and E47 are novel regulators of adiponectin.

Adiponectin is an adipocyte-derived cytokine with beneficial effects on insulin sensitivity and the development of atherosclerosis. Id3 is a helix-loop-helix factor that binds to E-proteins such as E47 and inhibits their binding to DNA. Although the helix-loop-helix factor sterol regulatory element binding protein (SREBP)-1c is a known activator of adiponectin transcription, this study provides the first evidence of a role for Id3 and E47 in adiponectin expression. Decreased Id3 in differentiating adipocytes correlates with increased adiponectin expression and forced expression of Id3 inhibits adiponectin expression. Moreover, Id3-null mice have increased adiponectin expression in visceral fat tissue and in serum. We demonstrate that E47 potentiates SREBP-1c-mediated adiponectin promoter activation and that Id3 can dose-dependently inhibit this action via interaction with E47. Mutation of a consensus E47 binding site results in nearly complete loss of promoter activation. Furthermore, we demonstrate E47 binding to the endogenous adiponectin promoter both in vitro and in vivo by chromatin immunoprecipitation analysis. Binding is not detected in undifferentiated cells which express Id3 but peaks during differentiation in parallel with Id3 decline. This promoter binding can be completely abolished by the overexpression of Id3 and is enhanced in adipose tissue null for Id3. These data establish Id3 and E47 as novel regulators of SREBP-1c-mediated adiponectin expression in differentiating adipocytes and provide evidence that Id3 regulates adiponectin expression in vivo.

Role of smooth muscle cells in the initiation and early progression of atherosclerosis.

The initiation of atherosclerosis results from complex interactions of circulating factors and various cell types in the vessel wall, including endothelial cells, lymphocytes, monocytes, and smooth muscle cells (SMCs). Recent reviews highlight the role of activated endothelium and inflammatory cell recruitment in the initiation of and progression of early atherosclerosis. Yet, human autopsy studies, in vitro mechanistic studies, and in vivo correlative data suggest an important role for SMCs in the initiation of atherosclerosis. SMCs are the major producers of extracellular matrix within the vessel wall and in response to atherogenic stimuli can modify the type of matrix proteins produced. In turn, the type of matrix present can affect the lipid content of the developing plaque and the proliferative index of the cells that are adherent to it. SMCs are also capable of functions typically attributed to other cell types. Like macrophages, SMCs can express a variety of receptors for lipid uptake and can form foam-like cells, thereby participating in the early accumulation of plaque lipid. Like endothelial cells, SMCs can also express a variety of adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 to which monocytes and lymphocytes can adhere and migrate into the vessel wall. In addition, through these adhesion molecules, SMCs can also stabilize these cells against apoptosis, thus contributing to the early cellularity of the lesion. Like many cells within the developing plaque, SMCs also produce many cytokines such as PDGF, transforming growth factor-beta, IFNgamma, and MCP-1, all of which contribute to the initiation and propagation of the inflammatory response to lipid. Recent advances in SMC-specific gene modulation have enhanced our ability to determine the role of SMCs in early atherogenesis.

Function of the N-terminus of zizimin1: autoinhibition and membrane targeting.

Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.

In vivo dynamics of Rac-membrane interactions.

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (k(off)) of membrane-bound GFP-Rac. We find that k(off) is 0.048 s(-1) for wtRac and approximately 10-fold less (0.004 s(-1)) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased k(off) for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on k(off). These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.

Identification of novel Smad2 and Smad3 associated proteins in response to TGF-beta1.

Transforming growth factor-beta 1 (TGF-beta1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. TGF-beta1 signals through the TGF-beta type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Since little is known about the selective activation of Smad2 versus Smad3, we set out to identify novel Smad2 and Smad3 interacting proteins in epithelial cells. A non-transformed human cell line was transduced with Myc-His(6)-Smad2 or Myc-His(6)-Smad3-expressing retrovirus and was treated with TGF-beta1. Myc-His(6)-Smad2 or Myc-His(6)-Smad3 was purified by tandem affinity purification, eluates were subject to SDS-PAGE and Colloidal Blue staining, and select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 or Smad3 were detected in the eluates. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified that have functions in cell proliferation, apoptosis, actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling. Specifically, the interaction between Smad2/3 and the Cdc42 guanine nucleotide exchange factor, Zizimin1, was validated by co-immunoprecipitation. The discovery of these novel Smad2 and/or Smad3 associated proteins may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-beta1 signaling.

Zizimin2: a novel, DOCK180-related Cdc42 guanine nucleotide exchange factor expressed predominantly in lymphocytes.

A novel superfamily of guanine nucleotide exchange factors for Rho GTPases includes DOCK180 and zizimin1. The zizimin subfamily includes three genes of which only zizimin1 has been cloned. We report here the cloning of zizimin2, identified in a screen for genes enriched in germinal center B cells. Zizimin2 and zizimin1 have similar primary structures and both proteins bound and activated Cdc42 but not the Cdc42-related proteins TC10 or TCL. Their tissue distributions are distinct, however, with zizimin2 expressed predominantly in lymphocytes and an opposite pattern for zizimin1. Zizimin3 was also analyzed and showed distinct GTPase specificity and tissue distribution.

Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness.

Kindlins are integrin-interacting proteins essential for integrin-mediated cell adhesiveness. In this study, we focused on the evolutionary origin and functional specialization of kindlins as a part of the evolutionary adaptation of cell adhesive machinery. Database searches revealed that many members of the integrin machinery (including talin and integrins) existed before kindlin emergence in evolution. Among the analyzed species, all metazoan lineages­but none of the premetazoans­had at least one kindlin-encoding gene, whereas talin was present in several premetazoan lineages. Kindlin appears to originate from a duplication of the sequence encoding the N-terminal fragment of talin (the talin head domain) with a subsequent insertion of the PH domain of separate origin. Sequence analysis identified a member of the actin filament-associated protein 1 (AFAP1) superfamily as the most likely origin of the kindlin PH domain. The functional divergence between kindlin paralogues was assessed using the sequence swap (chimera) approach. Comparison of kindlin 2 (K2)/kindlin 3 (K3) chimeras revealed that the F2 subdomain, in particular its C-terminal part, is crucial for the differential functional properties of K2 and K3. The presence of this segment enables K2 but not K3 to localize to focal adhesions. Sequence analysis of the C-terminal part of the F2 subdomain of K3 suggests that insertion of a variable glycine-rich sequence in vertebrates contributed to the loss of constitutive K3 targeting to focal adhesions. Thus emergence and subsequent functional specialization of kindlins allowed multicellular organisms to develop additional tissue-specific adaptations of cell adhesiveness.

Integrin ß3 crosstalk with VEGFR accommodating tyrosine phosphorylation as a regulatory switch.

Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between ß(3) integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of ß(3) and VEGFR2. Specifically, the membrane-proximal motif around (801)YLSI in VEGFR2 mediates its binding to non-phosphorylated ß(3)CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y(747) phosphorylation of ß(3) enhances the above interaction. To demonstrate the importance of ß(3) phosphorylation in endothelial cell functions, we synthesized ß(3)CT-mimicking Y(747) phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y(747) but not F(747) significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y(747) peptide exhibits inhibitory effect only in WT but not in ß(3) integrin knock-out or ß(3) integrin knock-in cells expressing ß(3) with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2.

CZH proteins: a new family of Rho-GEFs.

The Rho family of small GTPases are important regulators of multiple cellular activities and, most notably, reorganization of the actin cytoskeleton. Dbl-homology (DH)-domain-containing proteins are the classical guanine nucleotide exchange factors (GEFs) responsible for activation of Rho GTPases. However, members of a newly discovered family can also act as Rho-GEFs. These CZH proteins include: CDM (Ced-5, Dock180 and Myoblast city) proteins, which activate Rac; and zizimin proteins, which activate Cdc42. The family contains 11 mammalian proteins and has members in many other eukaryotes. The GEF activity is carried out by a novel, DH-unrelated domain named the DOCKER, CZH2 or DHR2 domain. CZH proteins have been implicated in cell migration, phagocytosis of apoptotic cells, T-cell activation and neurite outgrowth, and probably arose relatively early in eukaryotic evolution.

The novel Cdc42 guanine nucleotide exchange factor, zizimin1, dimerizes via the Cdc42-binding CZH2 domain.

Rho family small GTPases are critical regulators of multiple cellular processes and activities. Dbl homology domain-containing proteins are the classical guanine nucleotide exchange factors (GEFs) responsible for activation of Rho proteins. Recently another group of mammalian Rho-GEFs was discovered that includes CDM (Ced-5, DOCK180, Myoblast city) proteins that activate Rac and zizimin1 that activates Cdc42 via a nonconventional GEF module that we named the CZH2 domain. We report here that zizimin1 dimerizes via the CZH2 domain and that dimers are the only form detected. Dimerization was mapped to a approximately 200-amino acid region that overlaps but is distinct from the Cdc42-binding sequences. Rotary shadowing electron microscopy revealed zizimin1 to be a symmetric, V-shaped molecule. Experiments with DOCK180 and homology analysis suggest that dimerization may be a general feature of CZH proteins. Deletion and mutation analysis indicated existence of individual Cdc42-binding sites in the zizimin1 monomers. Kinetic measurements demonstrated increased binding affinity of Cdc42 to zizimin1 at higher Cdc42 concentration, suggesting positive cooperativity. These features are likely to be critical for Cdc42 activation.