HtrA1 Expression Research Articles

Hypoxia is involved in the reduction of HtrA3 in patients with endometrial hyperplasia and cancer

Endometrial cancer (EC) has recently become a major gynecological cancer and endometrial hyperplasia increases the risk for developing EC. Previous studies have reported that human high temperature requirement factor A3 (HtrA3), a member of ATP independent serine proteases family, is involved in endometrial carcinogenesis. However, the underlying mechanism of HtrA3 function is unclear in endometrial hyperplasia and cancer. In this study, we investigated that HtrA3 expression was reduced in endometrial hyperplasia as well as EC. The circulating levels of HtrA3 were also significantly reduced in both atypical hyperplasia and EC. Whether hypoxia is involved in the reduction of HtrA3 in EC was further investigated. Immunohistochemistry (IHC) scores of Glut1 and HtrA3 in type 1 and type 2 EC tissues showed the inverse correlation. And hypoxic condition reduced the expression of HtrA3. Furthermore, silencing HtrA3 promoted EC cell migration. Our study demonstrated the reduced levels of HtrA3 in endometrial hyperplasia including atypical hyperplasia which is a premalignant condition; and as the degree of hypoxia increases in EC, HtrA3 eventually loses its expression. Hypoxia is responsible for the reduction of HtrA3 which in turn promotes EC progression. These findings suggested that HtrA3 is an important adaptor in hypoxic regions that drives endometrial cancer development.

Elevated HTRA1 and HTRA4 in severe preeclampsia and their roles in trophoblast functions.

Aberrant gene expression during placental development may affect fetal growth and contribute to preeclampsia. The high‑temperature requirement A (HTRA) family of proteins are serine proteases that may serve in the quality control of misfolded or mislocalized proteins. Recently, the potential involvement of HTRA1 and HTRA4 in the normal development of the placenta and in the pathogenesis of preeclampsia has been reported. The present study collected placental tissues from patients with severe preeclampsia and gestational age‑matched control samples. The expression of HTRA1 and HTRA4 was analyzed using reverse transcription‑quantitative polymerase chain reaction, western blotting and immunohistochemistry. The human trophoblast line HTR‑8 was transfected with HTRA1 or HTRA4, and cell function was assessed. The present study also detected the expression of HTRA1 and HTRA4 in HTR‑8/SVneo transfected cells under hypoxia (1%O2) and further studied the effects of hypoxia on HTR‑8 cell migration. HTRA1 and HTRA4 were mainly localized to the cytoplasm of syncytiotrophoblasts. The expression levels of the two genes were elevated in the placental tissues of patients with severe preeclampsia. Finally, it was determined invitro that ectopic expression of HTRA1 and HTRA4 significantly attenuated HTR‑8 cell migration, and elevated HTRA1 limited HTR‑8 cell growth. Under hypoxic conditions, the expression levels of HTRA1 and HTRA4 improved significantly. It was hypothesized that the aberrant expression of HTRA1 or HTRA4 may be involved in the onset of preeclampsia, and increased HTRA1 or HTRA4 expression may affect trophoblast functions.

HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.

SummaryHigh‐temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age‐related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high‐risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high‐risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP‐1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD.

Inactivation of the serine protease HTRA1 inhibits tumor growth by deregulating angiogenesis.

The serine protease HTRA1 is involved in several vascular diseases and its expression is often deregulated in cancer. We aimed at identifying how HTRA1 in the vasculature affects tumor growth. Here we report that silencing of HTRA1 in cultured endothelial cells increased migration rate and tube formation, whereas forced HTRA1 expression impaired sprouting angiogenesis. Mechanistically, endothelial HTRA1 expression enhanced Delta/Notch signaling by reducing the amount of the weak Notch ligand JAG1. HTRA1 physically interacted with JAG1 and cleaved it within the intracellular domain, leading to protein degradation. Expression of a constitutive active Notch1 prevented the hypersprouting phenotype upon silencing of HTRA1. In HtrA1-deficient mice, endothelial Notch signaling was diminished and isolated endothelial cells had increased expression of VEGF receptor-2. Growth of syngeneic tumors was strongly impaired in HtrA1-/- mice. The tumor vasculature was much denser in HtrA1-/- mice and less covered with mural cells. This chaotic and immature vascular network was poorly functional as indicated by large hypoxic tumor areas and low tumor cell proliferation rates. In summary, inhibition of HTRA1 in the tumor stroma impaired tumor progression by deregulating angiogenesis.

High-temperature Requirement Protein A1 Regulates Odontoblastic Differentiation of Dental Pulp Cells via the Transforming Growth Factor Beta 1/Smad Signaling Pathway

IntroductionDentinogenesis includes odontoblast differentiation and extracellular matrix maturation as well as dentin mineralization. It is regulated by numerous molecules. High-temperature requirement protein A1 (HtrA1) plays crucial roles in bone mineralization and development and is closely associated with the transforming growth factor beta (TGF-β) signal in osteogenesis differentiation. Simultaneously, the TGF-β1/small mother against decapentaplegic (Smad) signaling pathway is an important signaling pathway in various physiological processes and as a downstream regulation factor of HtrA1. However, the role of HtrA1 and its relationship with the TGF-β1/Smad signaling pathway in dentin mineralization is unknown. MethodsWe detected the role of HtrA1 and its relationship with the TGF-β1/Smad signaling pathway in odontoblastic differentiation of human dental pulp cells (hDPCs) in this study. First, hDPCs were cultured in mineralized medium, and odontoblastic differentiation was confirmed by investigating mineralized nodule formation, alkaline phosphatase (ALP) activity, and the expression of mineral-associated genes, including ALP, collagen I, and dentin sialophosphoprotein. Then, the expression of HtrA1 and TGF-β1/Smad in hDPCs was investigated in hDPCs during mineralized induction. After HtrA1 knockdown by lentivirus, the mineralized nodule formation, ALP activity, and expression of mineral-associated genes and TGF-β1/Smad genes were investigated to confirm the effect of HtrA1 on odontoblastic differentiation and its relationship with the TGF-β1/Smad signaling pathway. ResultsThe expression of HtrA1 and TGF-β1 was increased during odontoblastic differentiation of hDPCs along with the messenger RNA expression of downstream factors of the TGF-β1/Smad signaling pathway. In addition, lentivirus-mediated HtrA1 knockdown inhibited the process of mineralization and the expression of HtrA1 and TGF-β1/Smad genes. ConclusionsThese findings suggest that HtrA1 might positively regulate odontoblastic differentiation of hDPCs through activation of the TGF-β1/Smad signaling pathway.

BMP-Responsive Protease HtrA1 Is Differentially Expressed in Astrocytes and Regulates Astrocytic Development and Injury Response

Astrocytes perform a wide array of physiological functions, including structural support, ion exchange, and neurotransmitter uptake. Despite this diversity, molecular markers that label subpopulations of astrocytes are limited, and mechanisms that generate distinct astrocyte subtypes remain unclear. Here we identified serine protease high temperature requirement A 1 (HtrA1), a bone morphogenetic protein 4 signaling regulated protein, as a novel marker of forebrain astrocytes, but not of neural stem cells, in adult mice of both sexes. Genetic deletion of HtrA1 during gliogenesis accelerates astrocyte differentiation. In addition, ablation of HtrA1 in cultured astrocytes leads to altered chondroitin sulfate proteoglycan expression and inhibition of neurite extension, along with elevated levels of transforming growth factor-β family proteins. Brain injury induces HtrA1 expression in reactive astrocytes, and loss of HtrA1 leads to an impairment in wound closure accompanied by increased proliferation of endothelial and immune cells. Our findings demonstrate that HtrA1 is differentially expressed in adult mouse forebrain astrocytes, and that HtrA1 plays important roles in astrocytic development and injury response.SIGNIFICANCE STATEMENT Astrocytes, an abundant cell type in the brain, perform a wide array of physiological functions. Although characterized as morphologically and functionally diverse, molecular markers that label astrocyte subtypes or signaling pathways that lead to their diversity remain limited. Here, after examining the expression profile of astrocytes generated in response to bone morphogenetic protein signaling, we identify high temperature requirement A 1 (HtrA1) as an astrocyte-specific marker that is differentially expressed in distinct adult mouse brain regions. HtrA1 is a serine protease that has been linked to cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, a small blood vessel disease in humans. Understanding the role of HtrA1 during development and after injury will provide insights into how distinct astrocyte populations are generated and their unique roles in injury and disease.

HtrA1 as a promising tissue marker in cancer: a meta-analysis

BackgroundHtrA1 is expressed in a variety of normal human tissues and seems to be involved in numerous physiological processes as well as tumorigenesis. This study reports the results of a meta-analysis that was performed: to compare HtrA1 expression as mRNA and protein, in cancer tissue versus non-cancer tissue and to assess overall survival in relation to low or medium-high HtrA1 tissue expression.MethodsThe PRISMA method was used for study selection. OR and HR with 95% confidence interval was used as a measure of effect size as appropriate. A random-effects model was applied to account for different sources of variation among studies. Heterogeneity across studies was assessed using Q statistic. Sensitivity analysis was conducted to check the stability of study findings. Egger’s regression method was applied to test funnel plot asymmetry.ResultsSensitivity analysis indicated the stability of meta-analytic findings in each meta-analysis. The study found a significantly different HtrA1 expression in cancer and non-cancer tissue. The meta-analysis of the prognostic studies showed a different survival according to HtrA1 expression.ConclusionsThe present data may provide a contribution to future work directed at exploring the role of HtrA1 in tumor development and progression and at establishing whether it may be used as a promising tissue marker for some tumors.

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index.

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-β1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

Paeoniflorin suppresses pancreatic cancer cell growth by upregulating HTRA3 expression.

Paeoniflorin (PF) is an active monoterpene glycoside extracted from Paeonia lactiflora Pall. PF has exhibited antitumor effects in various cancer types. However, the effects of PF in pancreatic cancer are largely unexplored. Here, we showed that PF suppressed growth of pancreatic cancer cell lines Capan-1 and MIAPaCa-2 and profoundly sensitized these cells to X-ray irradiation. Through microarray analysis, we identified HTRA3, a tumor-suppressor candidate gene, as the most increased gene upon PF treatment in Capan-1 cells. Ectopic expression of HTRA3 led to reduced cell proliferation and increased expression of apoptotic protein Bax, suggesting a tumor suppressive role of HTRA3 in pancreatic cancer cells. Together, our results provide a set group of genetic proofs and biological proofs that PF inhibited pancreatic cancer growth by upregulating HTRA3.

HtrA3 stromal expression is correlated with tumor budding in stage II colorectal cancer

Tumor budding is a well-established adverse prognostic factor in colorectal carcinoma (CRC). It may represent a form of epithelial-to-mesenchymal transition (EMT), although the underlying mechanisms remain unclear. High-temperature requirement A3 (HtrA3) is an inhibitor of the bone morphogenetic protein pathway, the suppression of which has been linked to EMT. Since HtrA3 is highly expressed in the desmoplastic stroma at the CRC invasive front, we sought to evaluate the relationship between tumor budding and HtrA3 expression in 172 stage II CRC resection specimens. All tumors were evaluated for tumor budding, with the highest budding slide selected for pan-keratin (CK) and HtrA3 immunohistochemistry. Representative areas of tumor core and invasive front, including budding and non-budding areas, were marked on CK stained slides, and then evaluated on HtrA3 stained slides. HtrA3 expression in tumor cells (tHtrA3) and peritumoral stroma (sHtrA3) was assessed for staining percentage and intensity (the product yielding a final score). Tumors with high-grade tumor budding (HGTB) showed increased expression of sHtrA3 in budding areas compared to non-budding areas at the invasive front (P<0.001). In addition, sHtrA3 expression at the invasive front was significantly higher in HGTB tumors compared to minimally budding tumors (P<0.05). tHtrA3 expression at the invasive front was significantly associated with high histological grade (P<0.05). Higher sHtrA3 expression in the tumor core (but not invasive front) was significantly associated with decreased 5-year overall survival on univariate analysis (P<0.05), but not multivariate analysis. HtrA3 expression in the peritumoral stroma of patients with stage II CRC is associated with HGTB and may be a novel marker of poor outcome.

Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-ß1

Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the association of 3D-culture in collagen sponges, under hypoxia in the presence of chondrogenic factors (BMP-2+TGF-β1) and siRNA to knockdown collagen I and HtrA1. Horse BM-MSCs were characterized before being cultured in chondrogenic conditions to find the best combination to enhance, stabilize, the chondrocyte phenotype. Our results show a very high proliferation of MSCs and these cells satisfy the criteria defining stem cells (pluripotency-surface markers expression). The combination of BMP-2+TGF-β1 strongly induces the chondrogenic differentiation of MSCs and prevents HtrA1 expression. siRNAs targeting Col1a1 and Htra1 were functionally validated. Ultimately, the combined use of specific culture conditions defined here with specific growth factors and a Col1a1 siRNAs (50nM) association leads to the in vitro synthesis of a hyaline-type neocartilage whose chondrocytes present an optimal phenotypic index similar to that of healthy, differentiated chondrocytes. Our results lead the way to setting up pre-clinical trials in horses to better understand the reaction of neocartilage substitute and to carry out a proof-of-concept of this therapeutic strategy on a large animal model.

Correlation Between Expression of High Temperature Requirement Serine Protease A1 (HtrA1) in Nucleus Pulposus and T2 Value of Magnetic Resonance Imaging.

BackgroundDegrading enzymes play an important role in the process of disc degeneration. The objective of this study was to investigate the correlation between the expression of high temperature requirement serine protease A1 (HtrA1) in the nucleus pulposus and the T2 value of the nucleus pulposus region in magnetic resonance imaging (MRI).Material/MethodsThirty-six patients who had undergone surgical excision of the nucleus pulposus were examined by MRI before surgery. Pfirrmann grading of the target intervertebral disc was performed according to the sagittal T2-weighted imaging, and the T2 value of the target nucleus pulposus was measured according to the median sagittal T2 mapping. The correlation between the Pfirrmann grade and the T2 value was analyzed. The expression of HtrA1 in the nucleus pulposus was analyzed by RT-PCR and Western blot. The correlation between the expression of HtrA1 and the T2 value was analyzed.ResultsThe T2 value of the nucleus pulposus region was 33.11–167.91 ms, with an average of 86.64±38.73 ms. According to Spearman correlation analysis, there was a rank correlation between T2 value and Pfirrmann grade (P<0.0001), and the correlation coefficient (rs)=−0.93617. There was a linear correlation between the mRNA level of HtrA1 and T2 value in nucleus pulposus tissues (a=3.88, b=−0.019, F=112.63, P<0.0001), normalized regression coefficient=−0.88. There was a linear correlation between the expression level of HtrA1 protein and the T2 value in the nucleus pulposus tissues (a=3.30, b=−0.016, F=93.15, P<0.0001) and normalized regression coefficient=−0.86.ConclusionsThe expression of HtrA1 was strongly related to the T2 value, suggesting that HtrA1 plays an important role in the pathological process of intervertebral disc degeneration.

Inhibition of cell proliferation, migration and apoptosis in blue-light illuminated human retinal pigment epithelium cells by down-regulation of HtrA1.

To investigate the effect of HtrA1 on the proliferation, migration and apoptosis of human retinal pigment epithelium (RPE) cells in the light injured model, as well as the expression of the apoptosis related molecules. The human RPE cell line ARPE-19 was exposed to blue light to establish the light injured model. The cells were transfected with HtrA1 siRNA to knockdown HtrA1 expression. Subsequent expression of HtrA1 was determined by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. Changes in cell proliferation, migration and apoptosis were assessed by cell counting kit-8 (CCK-8), Transwell assay and flow cytometry respectively, as well as changes in the mRNA and protein levels of Bax, Caspase-3 and Bcl-2 expression. HtrA1 was highly expressed in ARPE-19 cells after blue light irradiation. Knockdown of HtrA1 expression inhibited the proliferation, migration and apoptosis of the blue-light-irradiated ARPE-19 cells (P<0.05). Bax and Caspase-3 expression were significantly reduced both at mRNA and protein levels (P<0.05) after siRNA treatment. Bcl-2 expression significantly increased in blue-light-irradiated ARPE-19 cells after siRNA interference (P<0.05). Silence of HtrA1 may inhibit the proliferation, migration and apoptosis of ARPE-19 cells in light injured model. Moreover, HtrA1 suppression in blue-light-irradiated ARPE-19 cells may ameliorate cell apoptosis through down-regulation of Bax and Caspase-3, and up-regulation of Bcl-2 expression.

Effects of In Utero Thyroxine Exposure on Murine Cranial Suture Growth.

Large scale surveillance studies, case studies, as well as cohort studies have identified the influence of thyroid hormones on calvarial growth and development. Surveillance data suggests maternal thyroid disorders (hyperthyroidism, hypothyroidism with pharmacological replacement, and Maternal Graves Disease) are linked to as much as a 2.5 fold increased risk for craniosynostosis. Craniosynostosis is the premature fusion of one or more calvarial growth sites (sutures) prior to the completion of brain expansion. Thyroid hormones maintain proper bone mineral densities by interacting with growth hormone and aiding in the regulation of insulin like growth factors (IGFs). Disruption of this hormonal control of bone physiology may lead to altered bone dynamics thereby increasing the risk for craniosynostosis. In order to elucidate the effect of exogenous thyroxine exposure on cranial suture growth and morphology, wild type C57BL6 mouse litters were exposed to thyroxine in utero (control = no treatment; low ~167 ng per day; high ~667 ng per day). Thyroxine exposed mice demonstrated craniofacial dysmorphology (brachycranic). High dose exposed mice showed diminished area of the coronal and widening of the sagittal sutures indicative of premature fusion and compensatory growth. Presence of thyroid receptors was confirmed for the murine cranial suture and markers of proliferation and osteogenesis were increased in sutures from exposed mice. Increased Htra1 and Igf1 gene expression were found in sutures from high dose exposed individuals. Pathways related to the HTRA1/IGF axis, specifically Akt and Wnt, demonstrated evidence of increased activity. Overall our data suggest that maternal exogenous thyroxine exposure can drive calvarial growth alterations and altered suture morphology.

HTRA1 enhance signaling pathway of uveitis via modulation of the TGF-β signaling cascade

High temperature requirement protein A1 (HtrA1) is a serine protease that is mostly secreted to degrade numerous extracellular matrix proteins, They are involved in the development and progression of several pathological processes such as cancer, neurodegenerative disorders and arthritic diseases, but it also exists within cells for some partially understood. The purpose of this study was to investigate the role of HTRA1 in the uveitis, and the possible mechanisms involved. Interphotoreceptor retinoid-binding protein peptide R14 to induced uveitis in rat model. A recombinant lentiviral vector carrying HTRA1-shRNA to knockdown HTRA1 expression. ELISA to detected proinflammatory cytokines and standard molecular biological techniques were used for subcloning of HtrA1. The knockdown of HTRA1 was associated with decreased the cellularlevel expression of proinflammatory cytokine resulted in reduced cellular damage, and increased mRNA levels of TGF-β1.

Epigenetic silencing of serine protease HTRA1 drives polyploidy.

BackgroundIncreased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation.MethodsMouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated.ResultsHTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs).ConclusionsDownregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2425-8) contains supplementary material, which is available to authorized users.

HTRA1 promoter variant differentiates polypoidal choroidal vasculopathy from exudative age-related macular degeneration.

Exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) share similar abnormal choroidal vasculature, but responses to treatments are different. In this study, we sequenced the whole HTRA1 gene and its promoter by direct sequencing in a Hong Kong Chinese PCV cohort. We identified rs11200638, c.34delCinsTCCT, c.59C>T, rs1049331 and rs2293870 significantly associated with PCV. Notably, rs2672598 was significantly associated with exudative AMD (p = 1.31 × 10−4) than PCV (p = 0.11). Logistic regression indicated that rs2672598 (p = 2.27 × 10−3) remained significant after adjusting for rs11200638 in exudative AMD. Moreover, the rs11200638-rs2672598 joint genotype AA-CC conferred higher risk to exudative AMD (43.11 folds) than PCV (3.68 folds). Promoter analysis showed that rs2672598 C-allele showed higher luciferase expression than wildtype T-allele (p = 0.026), independent of rs11200638 genotype (p = 0.621). Coherently, vitreous humor HTRA1 expression with rs2672598 CC genotype was significantly higher than that with TT genotype by 2.56 folds (p = 0.02). Furthermore, rs2672598 C-allele was predicted to alter the transcription factor binding sites, but not rs11200638 A-allele. Our results revealed that HTRA1 rs2672598 is more significantly associated with exudative AMD than PCV in ARMS2/HTRA1 region, and it is responsible for elevated HTRA1 transcriptional activity and HTRA1 protein expression.

The expression of serine protease HtrA1 in human periodontal ligament tissue and the effect of HtrA1 on the proliferation of human periodontal ligament cells

To observe the expression of serine protease HtrA1 in human periodontal ligament tissue and to explore the effect of HtrA1 on the proliferation of human periodontal ligament cells (hPDLC). Six human premolars and three human third molars(patient's ages ranging from 12 to 25, with intact root, without caries and/or periodontitis) were obtained in the Department of Maxillofacial Surgery of Wuhan University Hospital of Stomatology. Reverse transcription-PCR(RT-PCR) and immunohistochemistry analysis were applied to investigate the expression of HtrA1. Primary hPDLC were obtained by tissue-culture method in vitro. The proliferation of hPDLC was determined by methyl thiazolytetrazolium(MTT). Lentivirus-mediated over-expression and reduction of HtrA1 level was performed. An empty vector was used as negative control. On days 1, 3, 5, 7 and 9, the growth of hPDLC was characterized using cell counting kit-8(CCK-8) assay. RT-PCR data indicated that HtrA1 mRNA was expressed in human periodontal ligament tissue. Immunohistochemistry analysis showed HtrA1 was expressed in human periodontal ligament, mainly in the cytoplasm of hPDLC and the extracellular matrix. The MTT result suggested that the growth curve was consistent with the growth characteristics of hPDLC. The stable over-expression and knockdown cell lines was successfully established by lentivirus with more than 90% transfection efficiency. CCK-8 assay showed that HtrA1 over-expression inhibited the proliferation of hPDLC(0.897±0.060, 0.890±0.083, 1.631±0.038, 1.111±0.041, 1.110±0.189), while cell proliferation increased after down-regulation of HtrA1(0.329±0.021, 0.529±0.044, 0.973±0.056, 1.626±0.102, 2.344±0.198)(P<0.05). HtrA1 is expressed in human periodontal ligament tissue at both mRNA and protein levels, and may play an important role in regulating the proliferation of hPDLC.

HtrA1 regulates epithelial–mesenchymal transition in hepatocellular carcinoma

Background and aimsEpithelial–mesenchymal transition (EMT) is involved in the development and progression of cancer. HtrA1 had been showed to play a modulatory role in metastasis of hepatocellular carcinoma (HCC). The relationship between HtrA1 and EMT in HCC was investigated in the present study. MethodsThe HtrA1 expression in human HCC tumor tissues and cells was determined by real-time PCR. SiRNA-HtrA1 and pcDNA-HtrA1 were respectively transfected into HepG2 and MHCC97H cells to observe their effects on cell migration and expression of EMT-associated markers Vimentin and E-cadherin. The relationship between HtrA1 and EMT in 60 HCC patients was also investigated. ResultsHtrA1 expression of tumor tissues was down-regulated with the increasing of number in lymph nodes metastasis in HCC patients. HtrA1 down-regulation led to the significant increase of cell migration, Vimentin expression and decrease of E-cadherin expression, while HtrA1 overexpression resulted in an opposite function. The HtrA1 expression was positively related to the E-cadherin level (R2 = 0.5903, P < 0.001) and negatively correlated with Vimentin level (R2 = 0.6067, P < 0.001) in tumor tissues of HCC, respectively. ConclusionHtrA1 expression was closely related to EMT, which might be a potential mechanism underlying metastasis of HCC.

Abstract 2385: Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HtrA1 and deregulated TGFB signaling

Abstract Dissemination of ovarian cancer is promoted by deregulated cell adhesion that selectively enhances detachment at primary site and attachment and invasion at secondary sites. Modifications in the extracellular matrix composition within tumor microenvironment can facilitate deregulated cell adhesion, migration, and invasion. We aim to understand how tumor-intrinsic changes affect expression and composition of ECM proteins in disseminated ovarian cancer. The serine protease HtrA1 has been shown to inhibit TGFB by targeting TGFB family ligands and receptors for proteolysis. In addition, HtrA1 also degrades ECM proteins, such as decorin, fibronectin, and collagens. We have shown that HtrA1 is down-regulated in ovarian cancer cells with loss contributing to chemotherapy resistance. We propose that loss of HtrA1 in the ovarian tumor microenvironment enhances TGFB signaling that in turn facilitate compositional changes in ECM proteins from stromal cells and promote cancer dissemination. To characterize compositional changes that occur in ECM proteins as a result of increased TGFB signaling, stable isotope amino acid labeling (SILAC) and LC-MS analysis were used. Mesothelial cells were cultured in heavy or light isotopically labeled media to confluence. Tumor cells expressing HtrA1 (NT) or stably transfected with HtrA1-targeting shRNA (sh1) were added to either heavy or light mesothelial cultures with or without TGFB (10 ng/ml). Cells were lifted by EDTA and the remaining matrix proteins were subjected to LC-MS. The data show that ECM compositional changes in fibronectin, laminin and collagen were up regulated in mesothelial cells co-cultured with sh1 compared to NT cells. All extracted proteins with ≥ 4 fold change were analyzed by Ingenuity Pathway Analysis. Proteins up regulated in co-cultures with HtrA1-deficient cancer cells (sh) were determined to be associated with migration, invasion and tumorogenesis whereas proteins upregulated in co-culture with HtrA1-expressing cancer cells (NT) were associated with protein modifications and ER stress. HtrA1-deficient cells show decreased adhesion to collagen I/III matrix and enhanced adhesion to collagen I/VI matrix. HtrA1-deficient cancer cells show morphology characteristics of epithelial-mesenchymal transition and shows higher motility and invasion through mesothelial cell layer. Finally, in clinical samples, decreased HtrA1 expression in primary tumor was significantly associated with higher incidence of carcinomatosis and nodal metastasis in ovarian cancer. We conclude that loss of HtrA1 in ovarian cancer cells results in compositional changes in ECM within the tumor microenvironment, and these changes may contribute to the dissemination of ovarian cancer. Citation Format: Megan Cooley, Jeremy Chien, Anirban Mitra, Alfonso Baldi, Andras Czirok, Edina Kosa. Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HtrA1 and deregulated TGFB signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2015-2385