Geun Bae Rha

The G0/G1 Switch Gene 2 Regulates Adipose Lipolysis through Association with Adipose Triglyceride Lipase

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for triacylglycerol (TAG) hydrolysis in adipocytes. The precise mechanisms whereby ATGL is regulated remain uncertain. Here, we demonstrate that a protein encoded by G(0)/G(1) switch gene 2 (G0S2) is a selective regulator of ATGL. G0S2 is highly expressed in adipose tissue and differentiated adipocytes. When overexpressed in HeLa cells, G0S2 localizes to lipid droplets and prevents their degradation mediated by ATGL. Moreover, G0S2 specifically interacts with ATGL through the hydrophobic domain of G0S2 and the patatin-like domain of ATGL. More importantly, interaction with G0S2 inhibits ATGL TAG hydrolase activity. Knockdown of endogenous G0S2 accelerates basal and stimulated lipolysis in adipocytes, whereas overexpression of G0S2 diminishes the rate of lipolysis in both adipocytes and adipose tissue explants. Thus, G0S2 functions to attenuate ATGL action both in vitro and in vivo and by this mechanism regulates TAG hydrolysis.

PPARα and PPARγ protect against HIV-1-induced MMP-9 overexpression via caveolae-associated ERK and Akt signaling

Activation of matrix metalloproteinase‐9 (MMP‐9) is involved in HIV‐1‐induced disruption of the blood‐brain barrier (BBB). In the present study, we hypothesize that peroxisome proliferator‐activated receptor (PPAR)‐α or PPARγ can protect against HIV‐1‐induced MMP‐9 overexpression in brain endothelial cells (hCMEC cell line) by attenuating cellular oxidative stress and down‐regulation of caveolae‐associated redox signaling. Exposure to HIV‐1‐infected monocytes induced phosphorylation of ERK1/2 and Akt in hCMEC by 2.5‐ and 3.6‐fold, respectively; however, these effects were attenuated by overexpression of PPARα or PPARγ and by silencing of caveolin‐1 (cav‐1). Coculture of hCMEC with HIV‐1‐infected monocytes significantly induced MMP‐9 promoter and enzyme activity by 3‐ to 3.5‐fold. Promoter mutation studies indicated that SP‐1 (g1940t_g1941t) is an essential transcription factor involved in induction of MMP‐9 promoter by HIV‐1. In addition, HIV‐1‐stimulated activity of MMP‐9 promoter was inhibited by mutation of AP‐1 site 2 (c1918t_a1919g) and both (but not individual) NF‐κB binding sites (g1389c and g1664c). PPAR overexpression, ERK1/2 or Akt inhibition, and silencing of cav‐1 all effectively protected against HIV‐1‐induced MMP‐9 promoter activity, indicating a close relationship among HIV‐1‐induced cerebrovascular toxicity, redox‐regulated mechanisms, and functional caveolae. Such a link was further confirmed in MMP‐9‐deficient mice exposed to PPARα or PPARγ agonist and injected with the HIV‐1‐specific protein Tat into cerebral vasculature. Overall, our results indicate that ERK1/2, Akt, and cav‐1 are involved in the regulatory mechanisms of PPAR‐mediated protection against HIV‐1‐induced MMP‐9 expression in brain endothelial cells.—Huang, W., András, I. E., Rha, G. B., Hennig, B., Toborek, M. PPARα and PPARγ protect against HIV‐1‐induced MMP‐9 overexpression via caveolae‐associated ERK and Akt signaling. FASEB J. 25, 3979–3988 (2011). www.fasebj.org

PPARα and PPARγ effectively protect against HIV-induced inflammatory responses in brain endothelial cells

Peroxisome proliferator‐activated receptors (PPARs) are nuclear receptors which down‐regulate inflammatory signaling pathways. Therefore, we hypothesized that alterations of PPAR functions can contribute to human immunodeficiency virus‐1 (HIV‐1)‐induced dysfunction of brain endothelial cells. Indeed, treatment with HIV‐1 transactivator of transcription (Tat) protein decreased PPAR transactivation in brain endothelial cells. We next stably over‐expressed PPARα and PPARγ in a newly developed cell line of human brain endothelial cells (hCMEC/D3 cells). Tat‐induced up‐regulation of inflammatory mediators, such as interleukin (IL)‐1β, tumor necrosis factor‐α, CCL2, and E‐selectin were markedly attenuated in hCMEC/D3 over‐expressing PPARα or PPARγ. These results were confirmed in CCL2 and E‐selectin promoter activity studies. Similar protective effects were observed in hCMEC/D3 after activation of PPARγ by exogenous PPAR agonists (dPGJ2 and rosiglitazone). PPAR over‐expression also prevented Tat‐induced binding activity and transactivation of nuclear factor‐κB. Importantly, increased PPAR activity attenuated induction of IL‐1β, tumor necrosis factor‐α, CCL2, and E‐selectin in hCMEC/D3 cells co‐cultured with HIV‐1‐infected Jurkat cells. The protective effects of PPAR over‐expression were reversed by the antagonists of PPARα (MK886) or PPARγ (GW9662). The present data suggest that targeting PPAR signaling may provide a novel therapeutic approach to attenuate HIV‐1‐induced local inflammatory responses in brain endothelial cells.

Structural Basis of Natural Promoter Recognition by a Unique Nuclear Receptor, HNF4α DIABETES GENE PRODUCT

HNF4α (hepatocyte nuclear factor 4α) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic β-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4α is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4α recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 Å crystal structure of human HNF4α DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1α, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4α molecular function can cause significant effects in afflicted MODY patients.

Inhibition of telomerase activity alters tight junction protein expression and induces transendothelial migration of HIV-1-infected cells

Telomerase, via its catalytic component telomerase reverse transcriptase (TERT), extends telomeres of eukaryotic chromosomes. The importance of this reaction is related to the fact that telomere shortening is a rate-limiting mechanism for human life span that induces cell senescence and contributes to the development of age-related pathologies. The aim of the present study was to evaluate whether the modulation of telomerase activity can influence human immunodeficiency virus type 1 (HIV-1)-mediated dysfunction of human brain endothelial cells (hCMEC/D3 cells) and transendothelial migration of HIV-1-infected cells. Telomerase activity was modulated in hCMEC/D3 cells via small interfering RNA-targeting human TERT (hTERT) or by using a specific pharmacological inhibitor of telomerase, TAG-6. The inhibition of hTERT resulted in the upregulation of HIV-1-induced overexpression of intercellular adhesion molecule-1 via the nuclear factor-kappaB-regulated mechanism and induced the transendothelial migration of HIV-1-infected monocytic U937 cells. In addition, the blocking of hTERT activity potentiated a HIV-induced downregulation of the expression of tight junction proteins. These results were confirmed in TERT-deficient mice injected with HIV-1-specific protein Tat into the cerebral vasculature. Further studies revealed that the upregulation of matrix metalloproteinase-9 is the underlying mechanisms of disruption of tight junction proteins in hCMEC/D3 cells with inhibited TERT and exposed to HIV-1. These results indicate that the senescence of brain endothelial cells may predispose to the HIV-induced upregulation of inflammatory mediators and the disruption of the barrier function at the level of the brain endothelium.

Multiple binding modes between HNF4alpha and the LXXLL motifs of PGC-1alpha lead to full activation.

Hepatocyte nuclear factor 4α (HNF4α) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4α interacts, peroxisome proliferation-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1α recruitment, we have determined the crystal structure of HNF4α in complex with a fragment of PGC-1α containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4α toward the LXXLL motifs of PGC-1α could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.

Probing the effect of MODY mutations near the co-activator-binding pocket of HNF4α.

HNF4α (hepatocyte nuclear factor 4α) is a culprit gene product for a monogenic and dominantly inherited form of diabetes, referred to as MODY (maturity onset diabetes of the young). As a member of the NR (nuclear receptor) superfamily, HNF4α recruits transcriptional co-activators such as SRC-1α (steroid receptor co-activator-1α) and PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) through the LXXLL-binding motifs for its transactivation, and our recent crystal structures of the complex provided the molecular details and the mechanistic insights into these co-activator recruitments. Several mutations have been identified from the MODY patients and, among these, point mutations can be very instructive site-specific measures of protein function and structure. Thus, in the present study, we probed the functional effects of the two MODY point mutations (D206Y and M364R) found directly near the LXXLL motif-binding site by conducting a series of experiments on their structural integrity and specific functional roles such as overall transcription, ligand selectivity, target gene recognition and co-activator recruitment. While the D206Y mutation has a subtle effect, the M364R mutation significantly impaired the overall transactivation by HNF4α. These functional disruptions are mainly due to their reduced ability to recruit co-activators and lowered protein stability (only with M364R mutation), while their DNA-binding activities and ligand selectivities are preserved. These results confirmed our structural predictions and proved that MODY mutations are loss-of-function mutations leading to impaired β-cell function. These findings should help target selective residues for correcting mutational defects or modulating the overall activity of HNF4α as a means of therapeutic intervention.