Induced recruitment and activation of Fyn kinase to the GHS-R1a receptor by ghrelin.

<p>(<b>A</b>) Ghrelin activates Src-related kinases. Differentiated THP-1 cells were serum-deprived for 16 h and incubated with 1 µM Src-related kinase inhibitor PP2 or 10 nM PI3-K inhibitor LY-294002 for 30 min before incubation with 10 nM ghrelin for the indicated times. The lysates were then analyzed by immunoblot with an anti-phospho-specific to Src Tyr-418, the activation loop site, and loading was monitored with a β-actin antibody. (<b>B</b>) Ghrelin promotes Fyn activation in THP-1 macrophages. Cells were treated as in (A) and extracts were immunoprecipitated with an antibody against Fyn, and analyzed by immunoblot using anti-Src-pY418 and anti-Fyn antibodies. (<b>C</b>) Fyn is recruited to the GHS-R1a receptor and activated by ghrelin. THP-1 cells were treated similar as in (A) and immunoprecipitation was carried out with an antibody against GHS-R1a, and analyzed by immunoblot using anti-Src-pY418 and anti-Fyn antibodies. Samples were normalized with an anti-GHS-R1a antibody. (<b>D</b>) Effect of GHS-R1a inhibition on Fyn recruitment and activation. Serum deprived THP-1 cells were pretreated with 100 nM d-Lys³-GHRP-6 for 30 min, followed by incubation with 10 nM ghrelin for 30 min, and then analyzed as in (C).</p

The ghrelin-induced PPARγ activation involves a negative regulation of Erk through Fyn/Dok-1 interaction.

<p>(<b>A</b>) Src inhibition increases Erk1/2 activity in differentiated THP-1 cells serum-deprived for 16 h and incubated with 1 µM PP2 for 30 min before incubation with 10 nM ghrelin for 2 to 30 min. The lysates were analyzed by immunoblot with antibodies specific to phospho-Erk1/2 and total Erk1/2. (<b>B</b>) Phosphorylation of PPARγ Ser-84 is increased by Src inhibition in THP-1 cells. Cells were treated as in (A), and lysates were immunoprecipitated with an antibody against PPARγ and analyzed by immunoblot using antibodies against phospho-serine, PPARγ pSer-84, and total PPARγ. (<b>C</b>) The PPARγ S84A mutant is insensitive to Src inhibition. 293 cells were transfected with UAStkLuc reporter in absence or presence of GHS-R1a, and with Gal4-PPARγ (<i>left</i>) or Gal4-PPARγS84A (<i>right</i>) plasmids. Cells were then treated with 10 nM ghrelin with or without 1 µM PP2 for 20 h, and harvested for luciferase assay. Normalized values are presented as fold changes ± SEM compared with untreated cells in presence of GHS-R1a. (<b>D</b>) Expression of Fyn inhibits Erk1/2 activity and PPARγ Ser-84 phosphorylation in the presence of ghrelin. 293 cells were transfected with GHS-R1a, Fyn and PPARγ expression plasmids, and then treated with 10 nM ghrelin for 5 and 15 min. Cell lysates were immunoblotted with the indicated antibodies. (<b>E</b>) Ghrelin promotes activation of Dok-1 and its recruitment to Fyn. 293 cells were transfected with Fyn and GHS-R1a expression plasmids in presence of Dok-1 or its inactive Y146F mutant, and treated with 10 nM ghrelin for the indicated time with or without 1 µM PP2. Immunoprecipitation was carried out with an antibody against HA and analyzed by immunoblot using anti-Src pTyr-418, anti-Fyn, anti-phospho-tyrosine (pTyr) and anti-Dok-1 antibodies. (<b>F</b>) Ghrelin promotes the recruitment of Dok-1 to Fyn in THP-1 macrophages. Cells were treated as in (A) and the lysates were immunoprecipitated with an antibody against Dok-1 and analyzed by immunoblot using the indicated antibodies.</p

Ghrelin induces PPARγ activation and phosphorylation independently of serine-84.

<p>(<b>A</b>) Ghrelin promotes PPARγ activity in 293 cells transfected with a UAStkLuc reporter plasmid in the presence of GHS-R1a and Gal4 fusion of full-length PPARγ1 expression plasmids. Transfected cells were treated with 10 nM ghrelin or 1.4 µM rosiglitazone for 20 h and harvested for luciferase activity. Values are normalized to β-galactosidase activity and expressed as fold changes in luciferase activity compared with untreated cells expressing GHS-R1a. Error bars represent the mean ±SEM of triplicate values derived from at least three independent experiments. (<b>B</b>) Similar as in (A) except that a truncated ABCDγ construct lacking the ligand binding domain, or a CDEFγ construct lacking the N-terminal region were used. (<b>C</b>) Activation of PPARγ by ghrelin is independent of serine 84. 293 cells were transfected as in (A) with an expression vector for Gal4-PPARγ or Gal4-PPARγS84A. Cells were treated with ghrelin as indicated or with 1.4 µM rosiglitazone for 20 h. (<b>D</b>) Phosphorylation of PPARγ in serum-deprived differentiated THP-1 cells treated with increasing doses of ghrelin for the indicated time. Cell extracts were immunoprecipitated with an antibody against PPARγ and analyzed by immunoblot using antibodies against phospho-serine residue or phospho-specific to PPARγ serine-84. Samples were normalized with a different anti-PPARγ antibody.</p

Ghrelin stimulates the PPARγ-LXRα-ABC pathway in THP-1 macrophages.

<p>(<b>A</b>) RT-PCR analysis of PMA-differentiated THP-1 macrophages treated or not with 100 nM ghrelin 48 h. Representative images are shown for the indicated genes. (<b>B</b>) Relative mRNA expression of THP-1 cells treated with increasing doses of ghrelin for 48 h. Rosiglitazone was used as a positive control of PPARγ activation. Data are presented as fold changes (±SEM) compared with undifferentiated cells, obtained from three to four separate experiments. (<b>C</b>) Representative immunoblot analysis of THP-1 cells treated with 10 nM ghrelin for 48 h. Relative fold changes are indicated. (D) PPARγ is required to mediate gene activation to ghrelin. Real-time gene expression analysis in THP-1 cells treated with 10 nM ghrelin for 48 h. PPARγ expression was silenced by infecting cells with a lentiviral-carrying shPPARγ and compared to a negative control shRNA. Results are normalized to GAPDH expression.</p

Activation of the PPARγ-LXRα-ABC transcriptional cascade by ghrelin is dependent on the PI3-K/Akt pathway.

<p>(<b>A</b>) Role of the PI3-K pathway on gene expression analysis from THP-1 cells treated with 100 nM ghrelin and 5 nM LY294002 for 48 h. Representative images and fold changes of mRNA expression are shown for the indicated genes. (<b>B</b>) Immunoblot analysis of THP-1 cells treated with ghrelin in presence or absence of 5 nM LY294002 for 48 h. Fold changes are shown relative to untreated cells. (<b>C</b>) A proposed model for the GHS-R1a-induced signaling to PPARγ in macrophages. Activation of GHS-R1a by ghrelin promotes the recruitment and activation of a Fyn/Dok-1 complex with the subsequent decrease in Erk1/2-mediated phosphorylation of PPARγ Ser-84, restraining its inhibitory potential. Ghrelin also activates the PI3-K/Akt pathway through a Gα_q–dependent mechanism, which then promotes PPARγ AF-1 phosphorylation independently of Ser-84, resulting in receptor transcriptional activation and increase in the PPARγ-LXRα-ABCA1/G1 metabolic cascade in macrophages.</p

Influences of Histidine‑1 and Azaphenylalanine‑4 on the Affinity, Anti-inflammatory, and Antiangiogenic Activities of Azapeptide Cluster of Differentiation 36 Receptor Modulators

Azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) exhibit promising affinity, selectivity, and modulator activity on the cluster of differentiation 36 receptor (CD36). For example, [A¹, azaF⁴]- and [azaY⁴]-GHRP-6 (<b>1a</b> and <b>2b</b>) were previously shown to bind selectively to CD36 and exhibited respectively significant antiangiogenic and slight angiogenic activities in a microvascular sprouting assay using choroid explants. The influences of the 1- and 4-position residues on the affinity, anti-inflammatory, and antiangiogenic activity of these azapeptides have now been studied in detail by the synthesis and analysis of a set of 25 analogues featuring Ala¹ or His¹ and a variety of aromatic side chains at the aza-amino acid residue in the 4-position. Although their binding affinities differed only by a factor of 17, the analogues exhibited significant differences in ability to modulate production of nitric oxide (NO) in macrophages and choroidal neovascularization

OS-Induced COX2 and VEGF Expression in RPE is CD36 Dependent

<div><p>(A–G) RT-PCR of cDNA from primary RPE cultures from Wistar rats and SHRs (A). Relative COX2 (B) and VEGF (G) mRNA expression (measured by real time RT-PCR. <i>n</i> = 6 wells per group; (B) *<i>p</i> = 0.0152 significant difference between control and CD36-deficient rats at 6 h; (G) *<i>p</i> = 0.0087 significant difference at 6 h) in RPE cells of Wistar (W) and SHR (S) rats exposed in culture to rod outer segments. COX2 (C and D) and VEGF (E and F) immunoreactivity (green) in 4-mo-old CD36^−/− (C and E) and CD36^+/+ (D and F) mice; tissues were counterstained with DAPI (nuclear stain).</p> <p>(H) Activation of CD36 with stimulating antibody evoked COX2 expression on RPE cell cultures from Wistar rats (W) and SHRs (measured by real time RT-PCR; <i>n</i> = 6 wells per group; *<i>p</i> = 0.0012 COX2 expression significantly different between control [Ctl] and antibody-treated [Ab] Wistar RPE culture).</p> <p>Photographs of immunohistochemical signal were taken with identical parameters in CD36^−/− and CD36^+/+ mice. Results are representative of at least three independent experiments. Ab, CD36 antibody FA6–152; CTL, control; RPE, retinal pigment epithelium. Scale bar: 50 μm</p></div

Choroidal Involution in COX2^−/− Mice

<div><p>(A and B) Micrographs of the retinal aspect of choriocapillaries in a frontal view of corrosion casts by scanning electron microscopy of 12-mo-old COX2^−/− (A) and COX2^+/+ (B) mice.</p> <p>(C–E) Quantification of the avascular area (<i>n</i> = 6 COX2^+/+ and <i>n</i> = 8 COX2^−/− eyes; *<i>p</i> = 0.007 COX2^−/− significantly different from COX2^+/+ at 12 mo) (C). Cross-sectional cuts of pericentral choroidal corrosion casts of COX2^−/− (D) and COX2^+/+ (E) mice.</p> <p>(F) Quantification of capillary thickness of 12-mo-old COX2^+/+ and COX2^−/− mice (<i>n</i> = 6 COX2^+/+ and <i>n</i> = 8 COX2^−/− eyes; *<i>p</i> = 0.0007).</p> <p>(G–I) Relative VEGF mRNA expression (by real time RT-PCR; <i>n</i> = 8 wells per group; *<i>p</i> = 0.0029 rod outer segments with DUP697 [R+D] significantly different from rod outer segments alone [R]) in primary RPE culture of Wistar rats (Ctl, white column), exposed to rod outer segments in absence (R, black column) or presence of the COX2 inhibitor DUP697 (10⁻⁶ M) (R+D, hatched column) (G). VEGF expression in 4-mo-old COX2^−/− (H) and COX2^+/+ (I) mice. Photomicrographs of immunohistochemical signal were taken with identical parameters.</p> <p>Results are representative of at least three independent experiments. m, months; RPE, retinal pigment epithelium. Scale bar: A, B, D, and E = 100 μm; H and I = 100 μm.</p></div

Retinal Degeneration in CD36-Deficient Animals

<div><p>(A) CD36 Western blot analysis of RPE/choroids complexes from Wistar rats (W) and SHRs S) (<i>n</i> = 4 eyes per group).</p> <p>(B and C) CD36 expression (green fluorescence) (B) and double-labeling with vascular marker BSA-1 (CD36 [green], BSA-1 [red], DAPI [blue]) (C) in CD36^+/+ mice (representative picture of three independent experiments).</p> <p>(D and E) Hemalun stained semi thin sections of 10-mo-old CD36-deficient SHRs (D) and control Wistar (W) rats (E).</p> <p>(F) Outer nuclear layer measurements of 10-mo-old Wistar rats (W; <i>n</i> = 6) SHRs (S; <i>n</i> = 8) (*<i>p</i> < 0.0001).</p> <p>(G and H) Hemalun-stained semi-thin sections (and periodic acid Schiff-stained paraffin sections [inset]) of 1-y-old CD36^−/− mice (G) and age-matched WT mice (H).</p> <p>(I) ONL thickness measurements in eyes of CD36^−/− (black bars; <i>n</i> = 10) and CD36^+/+ (white columns; <i>n</i> = 6) mice at different ages (*<i>p</i> = 0.0095 significant difference at 12 mo).</p> <p>(J–L) Transmission electron microscopy of the RPE and outer segments in SHRs (J), and CD36^−/− mice (K) and a CD36-expressing congener strain (CD36^+/+ mice) (L).</p> <p>Results are representative of at least three independent experiments. Scale bar: B, C, D, E, G, and H = 50 μm; J–L = 5μm.</p></div