Evolutionary History of the GABA Transporter (GAT) Group Revealed by Marine Invertebrate GAT-1

<div><p>The GABA transporter (GAT) group is one of the major subgroups in the solute career 6 (SLC6) family of transmembrane proteins. The GAT group, which has been well studied in mammals, has 6 known members, i.e., a taurine transporter (TAUT), four GABA transporters (GAT-1, -2, -3, - 4), and a creatine transporter (CT1), which have important roles in maintaining physiological homeostasis. However, the GAT group has not been extensively investigated in invertebrates; only TAUT has been reported in marine invertebrates such as bivalves and krills, and GAT-1 has been reported in several insect species and nematodes. Thus, it is unknown how transporters in the GAT group arose during the course of animal evolution. In this study, we cloned GAT-1 cDNAs from the deep-sea mussel, <i>Bathymodiolus septemdierum</i>, and the Antarctic krill, <i>Euphausia superba</i>, whose TAUT cDNA has already been cloned. To understand the evolutionary history of the GAT group, we conducted phylogenetic and synteny analyses on the GAT group transporters of vertebrates and invertebrates. Our findings suggest that transporters of the GAT group evolved through the following processes. First, GAT-1 and CT1 arose by tandem duplication of an ancestral transporter gene before the divergence of Deuterostomia and Protostomia; next, the TAUT gene arose and GAT-3 was formed by the tandem duplication of the TAUT gene; and finally, GAT-2 and GAT-4 evolved from a GAT-3 gene by chromosomal duplication in the ancestral vertebrates. Based on synteny and phylogenetic evidence, the present naming of the GAT group members does not accurately reflect the evolutionary relationships. </p> </div

Molecular phylogenetic tree of the GAT group members of vertebrates and invertebrates constructed by Bayesian method.

<p>The posterior probabilities are shown on the top left part of a branch. The scale bar represents a phylogenetic distance of 0.1 substitutions per site. <i>B. septemdierum</i> GAT-1 (BsGAT1) and <i>E. superba</i> GAT-1 (EsGAT1) are shown in red and their TAUTs are shown in blue. </p

Proposed molecular evolutionary history of GAT group member genes.

<p>* Since which of GAT-2 and GAT-4 was created first is unclear, their ancestor is shown as GAT-2/4. </p

Comparison of amino acid sequences of marine invertebrate and mammalian GAT-1.

<p>Human GAT-1 (HsGAT-1), ratGAT-1 (RnGAT-1), <i>B. septemdierum</i> GAT-1 (BsGAT1), and <i>E. superba</i> GAT-1 (EsGAT1) were compared. Identical amino acids are indicated by asterisks; conservative substitutions are indicated by a single dot; putative transmembrane domains (I-XII) are shaded; red boxes indicate position of amino acids known to be important for the functions of RnGAT-1.</p

Localization of GAT group member genes on chromosomes of vertebrates.

<p>(A) human, (B) green anole, (C) medaka. Ch, chromosome; LG, linkage group.</p

Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

<div><p>Tetraspanins organize protein complexes in tetraspanin-enriched membrane microdomains that are distinct from lipid rafts. Our previous studies suggested that reduction in the levels of tetraspanins CD9 and CD81 may be involved in the progression of inflammatory lung diseases, especially COPD. To search for agents that increase the levels of these tetraspanins, we screened 1,165 drugs in clinical use and found that statins upregulate CD9 and CD81 in RAW264.7 macrophages. The lipophilic statins, fluvastatin and simvastatin, reversed LPS-induced downregulation of CD9 and CD81, simultaneously preventing TNF-α and matrix metalloproteinase-9 production and spreading of RAW264.7 cells. These statins exerted anti-inflammatory effects <i>in vitro</i> in wild-type macrophages but not in CD9 knockout macrophages, and decreased lung inflammation <i>in vivo</i> in wild-type mice but not in CD9 knockout mice, suggesting that their effects are dependent on CD9. Mechanistically, the statins promoted reverse transfer of the LPS-signaling mediator CD14 from lipid rafts into CD9-enriched microdomains, thereby preventing LPS receptor formation. Finally, upregulation of CD9/CD81 by statins was related to blockade of GTPase geranylgeranylation in the mevalonate pathway. Our data underscore the importance of the negative regulator CD9 in lung inflammation, and suggest that statins exert anti-inflammatory effects by upregulating tetraspanin CD9 in macrophages.</p> </div

Statins protect mice from LPS-induced injury in a CD9-dependent manner.

<p>(<b>A</b>) WT mice were repeatedly intraperitoneally injected with vehicle (-) or 30 mg/kg fluvastatin (Fluv), and unchallenged (-) or intraperitoneally challenged with 30 mg/kg LPS (+). After 48 h, BMDMs were isolated and the level of CD9 was examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. (<b>B</b>) WT mice were treated as in <b>A</b>. <i>CD9</i> mRNA levels in the BMDMs were examined by reverse transcription PCR. <i>GAPDH</i> is an internal loading control. (<b>C</b>) WT and CD9 KO mice were repeatedly intraperitoneally injected with vehicle (-) or 30 mg/kg fluvastatin (+), and intranasally challenged with 0.5 mg/kg LPS (+). After 24 h, activities of MMP-2 and MMP-9 in BALF were analyzed by gelatin zymography. (<b>D</b>) WT and CD9 KO mice were untreated (Cont, control) or intraperitoneally injected with vehicle or 20 mg/kg simvastatin (Simv) and intranasally challenged with 0.5 mg/kg LPS. After 4 days, total cells in BALF from the mice from each group (<i>n</i> = 9) were counted using a hemocytometer. Each bar represents the mean ± SEM. (<b>E</b>) WT and CD9 KO mice were treated as in <b>D</b>. Histological lung sections collected at 4 days were stained with hematoxylin and eosin. Scale bar, 100 µm. (<b>F</b>) WT and CD9 KO mice were intraperitoneally injected with vehicle or 20 mg/kg simvastatin, and intraperitoneally challenged with 40 mg/kg LPS. Survival of the mice from each group (<i>n</i> = 12) was monitored and analyzed by the Kaplan-Meier method. ^⋆<i>P</i> < 0.05; ^{⋆ ⋆}<i>P</i> < 0.01.</p

Statins transfer CD14 from lipid rafts into CD9-enriched microdomains.

<p>(<b>A</b>) RAW264.7 cells were stimulated with 0.1 µg/ml LPS and, after the indicated times, the cells were lysed and protein levels were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. (<b>B</b>) RAW264.7 cells were untreated (-) or cultured for 24 h in the absence (-) or presence of 5 µM fluvastatin (Fluv) or simvastatin (Simv) (+) and stimulated for 2 h with 1 µg/ml LPS (+). Proteins in whole-cell lysate (WCL) and CD14 protein in immunoprecipitates (IP) with anti-TLR4 Ab were immunoblotted (IB). (<b>C</b>) RAW264.7 cells were treated as in <b>B</b>. Lysates of untreated (C, control) cultures or LPS-stimulated cultures in the absence (L) or presence of fluvastatin (FL) or simvastatin (SL) were fractionated by sucrose density gradients, and protein distributions were visualized by immunoblotting. The intensities of blots were quantified by densitometry, and percentages of density units of light membrane (LM) fractions are displayed to the right of the blots. Data shown are from one representative of three similar experiments. (<b>D</b>) Immunoblots of CD9 and CD81 proteins in whole-cell lysates and in immunoprecipitates with control IgG or anti-CD14 mAb. (<b>E</b>) Immunoblots of CD9 and CD81 proteins in whole-cell lysates and in immunoprecipitates with control IgG or anti-CD14 mAb from pooled LM fractions (4 and 5) and dense (D) fractions (9 and 10). In the presence of statins, more CD14/CD9 complexes were formed in dense fractions (arrowheads).</p

Fluvastatin and simvastatin increase CD9 and CD81 levels in RAW264.7 cells.

<p>(<b>A</b>) RAW264.7 cells were cultured for 24 h in the absence or presence of increasing concentrations of fluvastatin (Fluv) or simvastatin (Simv). The cells were lysed, and levels of CD9, CD63, and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. (<b>B</b>) RAW264.7 cells were untreated (-) or cultured in the absence or presence of increasing concentrations of fluvastatin or simvastatin and stimulated for 24 h with 0.1 µg/ml LPS (+). Levels of CD9, CD63, and CD81 were examined by immunoblotting. Note that LPS downregulates CD9 and CD81 in the absence of statins (arrowheads). (<b>C</b>) RAW264.7 cells were cultured in the absence (-) or presence of 3 µM fluvastatin (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). mRNA levels of <i>CD9</i> and <i>CD81</i> were examined by reverse transcription PCR. <i>GAPDH</i> is an internal loading control. (<b>D</b>) RAW264.7 cells were cultured in the absence or presence of fluvastatin, and unstimulated or stimulated with LPS. Control (Cont) was an untreated culture. mRNA levels of <i>CD9</i> and <i>CD81</i> were examined by real-time PCR. Data shown are from one representative of three similar experiments. (<b>E</b>) Human monocytic THP-1 cells were treated for 4 h with 1 µg/ml phorbol 12-myristate 13-acetate, allowed to attach to a plate, and then cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting. (<b>F</b>) Mouse 3T3 fibroblasts were cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting.</p

Blockade of the mevalonate pathway increases CD9 and CD81.

<p>(<b>A</b>) RAW264.7 cells were untreated (-) or treated for 48 h with 50 ng/ml TSA (+) in the absence (-) or presence of 50 µM theophylline or 0.5 µM fluvastatin (Fluv) (+). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. (<b>B</b>) The mevalonate pathway and inhibitors. n-BP, nitrogenous bisphosphonate. (<b>C</b>) RAW264.7 cells were cultured for 24 h in the presence of indicated concentrations of fluvastatin, simvastatin (Simv), zoledronate (Zol), or risedronate (Ris). Levels of CD9 and CD81 were examined by immunoblotting. (<b>D</b>) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) or presence of mevalonate (Mev), farnesyl pyrophosphate (FPP), squalene (Squ), or geranylgeranyl pyrophosphate (GGPP). Although the actin level in the GGPP lane appears to be lower, an equal amount of protein was loaded. (<b>E</b>) RAW264.7 cells were cultured for 24 h in the absence (V) or presence of fluvastatin, zoledronate, farnesyl transferase inhibitor (FTI), or geranylgeranyl transferase inhibitor (GGTI). (<b>F</b>) RAW264.7 cells were untreated (-) or treated with fluvastatin (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP. (<b>G</b>) RAW264.7 cells were untreated (-) or treated with zoledronate (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP. (<b>H</b>) RAW264.7 cells were untreated (-) or treated with fluvastatin (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP and stimulated for 15 min with 0.1 µg/ml LPS (+). The cells were lysed, and levels of IκBα were examined by immunoblotting. (<b>I</b>) RAW264.7 cells were cultured for 24 h in the indicated concentrations of HA1077. Levels of CD9 and CD81 were examined by immunoblotting.</p