Fig 1: Comparison of Htra1 expression and secretion in mouse and human.A, quantitative real-time PCR determination of Htra1 mRNA levels from total RNA isolated from the brains, retina, liver, lung, kidney, and blood of WT and Htra1 Tg mice. Htra1 is expressed at a low level but ubiquitously in all tissues analyzed. In comparison with WT, Htra1 Tg mouse expressed 49.7-, 30-, and 3.4-fold in the retina, blood and brain, respectively. The results are expressed as the mean ± SEM. The p value obtained by Student's t test, n = 3. B, ELISA analysis of Htra1 expression in mouse serum. The Htra1 protein level is significantly higher in Htra1 Tg mice than in WT mice (p = 0.0385, n = 16). Data are presented as the mean ± SEM. The p value obtained by two-tailed Mann–Whitney is indicated above the graph. C, the HTRA1 protein concentration in plasma or serum was determined by ELISA and it was significantly enhanced in AMD cases comparing with controls in Japanese samples (plasma, **p = 0.0016; serum, *p = 0.0362), Indian samples (plasma, **p = 0.0035; serum, ****p < 0.0001), Australian samples (plasma, **p = 0.0039), samples of USA (serum, *p = 0.0293). Data are presented as the mean ± SEM. The p value obtained by the two-tailed Mann–Whitney test is indicated above each graph. D, In/del variant significantly effects HTRA1 concentration in Japanese samples (plasma, ***p < 0.001; serum, ***p < 0.001), Indian samples (plasma, ***p < 0.001; serum, ***p < 0.001), Australian samples (plasma, ***p < 0.001), and samples of USA (serum, ***p < 0.001) (Table S3). Data are presented as the mean ± SEM. p values were derived by two-way ANOVA, with Tukey's multiple comparisons test. The p (>0.05) value is indicated above each graph.
Fig 2: Action of HTRA1 on TGF-ß/ALK5/SMAD2/3 signaling.A, we assayed the effects of HTRA1 on pSMAD2/3, SMAD2/2, VEGF, and TGFßRII by transfecting pCMV-Myc-HTRA1 vector in HEK-293, 661W and HeLa cells, followed by WB. Addition of the HTRA1 expression vector significantly inhibited SAMD2/3 phosphorylation but not SMAD 2/3 expression in HEK-293 cells. HTRA1 enhanced VEGF expression in HeLa cells and cleavage TGFßRII in all three cell lines. B, the band intensity was analyzed by Image J software. C, influence of overexpressed HTRA1 in mice. Phospho-Smad2/3 and TGFßRII, but not Smad2/3, decreased in 1-year-old Htra1 Tg mouse compared with WT mouse. VEGF enhanced in Htra1 Tg mouse compared with WT mouse. D, analysis of band intensities. E, mRNA levels of TGF II, TGFßRII, and ALK5 in HTRA1 transfected HEK-293, 661W, and HeLa cells, respectively. qRT-PCR analysis of TGF II, TGFßRII, and Alk5 mRNA levels (F) and VEGF isoforms (VEGF120, VEGF164, and VEGF188) (G) in the retina of Htra1 Tg mouse. The expression of VEGF120 isoform mRNA was significantly enhanced in 1-year-old Htra1 Tg mouse compared with WT. The other two VEGF isoforms were undetectable. Throughout, the results are expressed as the mean ± SEM. The p value was obtained by Student's t test.
Fig 3: In/del binding transcription factor protein. A, schematic illustration of the double-stranded DNA probes for EMSA (gel electrophoresis mobility shift assay) in the region upstream of the HTRA1 activity coding region. Linking c-fos transcription factor to in/del-6. Profile of the c-fos transcription factor-binding sequence (ID: MA0099.3 from the JASPAR database). B, EMSA was performed to determine the in/del binding activity protein. In total, 50 µl nuclear protein from 661W cells was incubated with 100 pmol biotin-labeled Double-stranded DNA probes and analyzed on a 7.5% EMSA gel. Bands of interest were cut out and processed for LC-MS/MS analysis. C, Gtf2i-DNA probes binding test. The binding ability between Gtf2i and the in/del region was confirmed by WB. In/del DNA probes 6 and 7 were detected by an anti-Gtf2i antibody. Detection of Lamin A/C was used as an internal control. D, gene structure of Murine Gtf2i. Coding exons are depicted as black boxes and noncoding exons are in blank boxes. Five isoforms signify various alternatively spliced isoforms with exon 9, 10, 11, and 12 indicated. In 661W cells, there are only isoforms ß and d present by TA cloning (E, F). G, Gtf2i ß/d -DNA probes binding test. Both Gtf2i ß and d bind to the in/del-6 probe by WB. H, expression of Gtf2i isoform ß and d in nuclear or cytoplasmic extracts. Vector of Gtf2i isoform ß or d was transfected into 661W cells, followed by WB. Gtf2i isoform ß was expressed in both nuclear and cytoplasmic extracts. However, isoform d was only detected in nuclear but cytoplasmic extracts.
Fig 4: Function analysis for Gtf2i and HTRA1.A, HTRA1 in/del and normal with or without Gtf2i ß/d vectors were transfected in HEK-293, 661W and COS-7 cells, respectively, following ELISA. Presence of the in/del significantly enhanced HTRA1 secretion and Gtf2i ß/d promoted enhanced efficiency with indel-HTRA1 but not the normal-HTRA1 in all cell lines. B, secretion of HTRA1 in human iPSCs derived from AMD patients. iPSCs were derived from individuals with normal versus in/del transcription regulators, followed by ELISA. Transfection of 661W cells with the Gtf2i ß or d expression vectors significantly enhanced in/del-Luciferase transcription comparing with normal. Representative WB (C) and immunocytochemistry (D) results are shown. Throughout, the results are expressed as the mean ± SEM. The p value was obtained by Student's t test.
Fig 5: Correlation analysis of age and blood level of HTRA1. HTRA1 increased progressively with age in all controls. However, in AMD cases from each country, no correlation was found between blood concentration of HTRA1 and age. Data are presented as scatter plots. The linear regression equations are solid, straight lines. The correlation coefficient (r) and p (two-tailed) values were obtained using linear regression (Pearson's) analysis.
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