HtrA1 Expression Research Articles

Fetal growth restriction caused by MIMT1 deletion alters brain transcriptome in cattle

We examined levels of gene expression in the brains of bovine fetuses carrying a truncated MIMT1 allele, MIMT1Del, shown to cause late abortion and stillbirth as a result of fetal growth restriction. MIMT1 is a non-protein coding gene that forms part of the imprinted PEG3 (paternally expressed gene 3) domain. Microarray analysis of brain cortex samples from mid-gestation MIMT1Del/WT bovine fetuses and wild-type siblings was performed to study the effect of fetal growth restriction on brain gene expression. Statistical analysis revealed 134 genes with increased mRNA levels and 22 with reduced levels in MIMT1Del/WT fetuses. Gene set enrichment analysis identified a relatively small number of significant functional clusters representing three major biological processes: response to oxidative stress, angiogenesis, and epithelial cell proliferation. Gene expression microarray analyses identified increased expression of VIPR2, HTRA1, S100A4 and MYH8 in fetuses carrying the deletion and decreased expression of DRD2, ADAM18, miR345, ZNF585A. ADAM18, DRD2 and S100A4 are known to be involved in prenatal brain development. ZNF585A, miR-345, VIPR2, HTRA1, and MYH8 are known to be involved in cell growth and differentiation, but any role in neural developmental has yet to be elucidated.

Variants at chromosome 10q26 locus and the expression of HTRA1 in the retina

Variations in a locus at chromosome 10q26 are strongly associated with the risk of age-related macular degeneration (AMD). The most significantly associated haplotype includes a nonsynonymous SNP rs10490924 in the exon 1 of ARMS2 and rs11200638 in the promoter region of HTRA1. It is under debate which gene(s), ARMS2, HTRA1 or some other genes are functionally responsible for the genetic association. To verify whether the associated variants correlate with a higher HTRA1 expression level as previously reported, HTRA1 mRNA and protein were measured in a larger human retina-RPE-choroid samples (n = 82). Results show there is no significant change of HTRA1 mRNA level among genotypes at rs11200638, rs10490924 or an indel variant of ARMS2. Furthermore, two AMD-associated synonymous SNPs rs1049331 and rs2293870 in HTRA1 exon 1 do not change its protein level either. These results suggest that the AMD-associated variants in the chromosome 10q26 locus do not significantly affect the expression of HTRA1.

HtrA1 is induced by oxidative stress and enhances cell senescence through p38 MAPK pathway

Genetic predisposition and senescence of retinal pigment epithelium induced by oxidative stress are major contributors to age-related macular degeneration (AMD). Single-nucleotide polymorphisms in HTRA1 are strongly linked to the onset of AMD. In this study, we examine the role of HtrA1 in premature senescence and cell death induced by oxidative stress. HtrA1 mRNA and protein were up-regulated during premature senescence induced by H2O2 in both mouse embryonic fibroblasts (MEFs) and ARPE-19 cells. Expression of the senescence markers p21CIP1/WAF1 and p16INK4a, and SA-β-galactosidase activity, were higher in HtrA1+/− MEFs than in HtrA1−/− MEFs. HtrA1+/+ and HtrA1+/− MEFs were more resistant than HtrA1−/− MEFs to H2O2-induced cell death. Activation of p38 MAPK by oxidative stress was quicker in HtrA1+/− MEFs than in HtrA1−/− MEFs. The effects of excess HtrA1 were examined by transient transfection of cells with HtrA1 expression vectors or by addition of recombinant proteins. Excess wild type HtrA1 accelerated premature senescence of MEFs and ARPE-19 cells, while the protease-inactive HtrA1 S328A did not. HtrA1-induced senescence was abrogated by inhibition of p38 MAPK. We conclude that HtrA1 is induced by oxidative stress and promotes premature cell senescence through p38 MAPK in a protease activity-dependent manner.

Downregulation of Serine Protease HTRA1 Is Associated with Poor Survival in Breast Cancer

HTRA1 is a highly conserved serine protease which has been implicated in suppression of epithelial-to-mesenchymal-transition (EMT) and cell motility in breast cancer. Its prognostic relevance for breast cancer is unclear so far. Therefore, we evaluated the impact of HTRA1 mRNA expression on patient outcome using a cohort of 131 breast cancer patients as well as a validation cohort including 2809 publically available data sets. Additionally, we aimed at investigating for the presence of promoter hypermethylation as a mechanism for silencing the HTRA1 gene in breast tumors. HTRA1 downregulation was detected in more than 50% of the breast cancer specimens and was associated with higher tumor stage (p = 0.025). By applying Cox proportional hazard models, we observed favorable overall (OS) and disease-free survival (DFS) related to high HTRA1 expression (HR = 0.45 [CI 0.23–0.90], p = 0.023; HR = 0.55 [CI 0.32–0.94], p = 0.028, respectively), with even more pronounced impact in node-positive patients (HR = 0.21 [CI 0.07–0.63], p = 0.006; HR = 0.29 [CI 0.13–0.65], p = 0.002, respectively). Moreover, HTRA1 remained a statistically significant factor predicting DFS among established clinical parameters in the multivariable analysis. Its impact on patient outcome was independently confirmed in the validation set (for relapse-free survival (n = 2809): HR = 0.79 [CI 0.7–0.9], log-rank p = 0.0003; for OS (n = 971): HR = 0.63 [CI 0.48–0.83], log-rank p = 0.0009). In promoter analyses, we in fact detected methylation of HTRA1 in a small subset of breast cancer specimens (two out of a series of 12), and in MCF-7 breast cancer cells which exhibited 22-fold lower HTRA1 mRNA expression levels compared to unmethylated MDA-MB-231 cells. In conclusion, we show that downregulation of HTRA1 is associated with shorter patient survival, particularly in node-positive breast cancer. Since HTRA1 loss was demonstrated to induce EMT and cancer cell invasion, these patients might benefit from demethylating agents or histone deacetylase inhibitors previously reported to lead to HTRA1 upregulation, or from novel small-molecule inhibitors targeting EMT-related processes.

Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

Multiple genetic studies have suggested that high-temperature requirement serine protease (HTRA1) is associated with polypoidal choroidal vasculopathy (PCV). To date, no functional studies have investigated the biological effect of HTRA1 on vascular endothelial cells, essential vascular components involved in polypoidal vascular abnormalities and arteriosclerosis-like changes. In vitro studies were performed to investigate the effect of HTRA1 on the regulation of fibronectin, laminin, vascular endothelial growth factor (VEGF), platelet derived growth factor receptor (PDGFR) and matrix metalloparoteinases 2 (MMP-2) and the role of HTRA1 in choroid-retina endothelial (RF/6A) and human umbilical vein endothelial (HUVEC) cells. Lentivirus-mediated overexpression of HTRA1 was used to explore effects of the protease on RF/6A and HUVEC cells in vitro. HTRA1 overexpression inhibited the proliferation, cell cycle, migration and tube formation of RF/6A and HUVEC cells, effects that might contribute to the early stage of PCV pathological lesions. Fibronectin mRNA and protein levels were significantly down-regulated following the upregulation of HTRA1, whereas the expressions of laminin, VEGF and MMP-2 were unaffected by alterations in HTRA1 expression. The decreased biological function of vascular endothelial cells and the degradation of extracellular matrix proteins, such as fibronectin, may be involved in a contributory role for HTRA1 in PCV pathogenesis.

HtrA1 expression associated with the occurrence and development of esophageal cancer.

BackgroundThe purposes of this study were to measure both the mRNA and protein expression levels of high-temperature requirement serine peptidase 1 (HtrA1) in human esophageal cancer tissues and their adjacent, comparatively normal esophageal tissues.MethodsThe expression levels of HtrA1 mRNA and protein in both tissue types were measured by semi-quantitative RT-PCR (reverse transcription-polymerase chain reaction) and Western blotting. The clinical and pathological correlation between HtrA1 expression levels and the occurrence and development of esophageal cancer was analyzed.ResultsThe expression levels of HtrA1 mRNA and protein in esophageal carcinoma were significantly lower than the levels expressed in their adjacent normal esophageal tissue (p < 0.05). The more highly undifferentiated esophageal tumor cells expressed lower HtrA1 mRNA and protein expression levels (p < 0.05). Patients with tumors in early pathological stages (I-II) had significantly higher HtrA1 mRNA and protein expression levels than did patients with tumors in mid-to-late pathological stages (III-IV) (p < 0.05). Patients with positive lymph node metastasis had significantly lower HtrA1 mRNA and protein expression levels than did patients with lymph node-negative disease (p < 0.05).ConclusionsHtrA1 expression is associated with the occurrence and development of esophageal cancer.

Changes in expression of human serine protease HtrA1, HtrA2 and HtrA3 genes in benign and malignant thyroid tumors

Human HtrA proteins are serine proteases involved in essential physiological processes. HtrA1 and HtrA3 function as tumor suppressors and inhibitors of the TGF-β signaling pathway. HtrA2 regulates mitochondrial homeostasis and plays a pivotal role in the induction of apoptosis. The aim of the study was to determine whether the HtrA proteins are involved in thyroid carcinogenesis. We used the immunoblotting technique to estimate protein levels of HtrA1, HtrA2, long and short variants of HtrA3 (HtrA3-L and HtrA3-S) and TGF-β1 in tissues of benign and malignant thyroid lesions, and control groups. We found that the levels of HtrA2 and HtrA3-S were higher in thyroid malignant tumors compared to normal tissues and benign tumors. The HtrA3-L level was increased in malignant tumor tissues compared to benign tumor tissues and control tissues from patients with benign lesions, and elevated in normal tissues from patients with thyroid carcinoma compared to normal tissues from patients with benign lesions. We also compared levels of HtrA proteins in follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC) and found that these types of carcinoma differed in the expression of HtrA3-S and HtrA1. These results indicate the implication of HtrA proteins in thyroid carcinogenesis suggest that HtrA3 variants may play different roles in cancer development, and that the increased HtrA3-L levels in thyroid tissue could be correlated with the development of malignant lesions. The TGF-β1 levels in tumor tissues were not significantly altered compared to control tissues.

Down-Regulation of HtrA1 Activates the Epithelial-Mesenchymal Transition and ATM DNA Damage Response Pathways

Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.

A new idea for simple and rapid monitoring of gene expression: requirement of nucleotide sequences encoding an N-terminal HA tag in the T7 promoter-driven expression in E. coli

Mammalian expression vectors are used to overexpress genes of interest in mammalian cells. High temperature requirement protein A1 (HtrA1), used as a specific target, was expressed from the pHA-M-HtrA1 plasmid in HEK293T cells, inducing cell death. Expression of HtrA1 was driven by the pHA-M-HtrA1 mammalian expression vector in E. coli resulting in growth suppression of E. coli in an HtrA1 serine protease-dependent manner. By using various combinations of promoters, target genes and N-terminal tags, the T7 promoter and N-terminal HA tag in the mammalian expression vector were shown to be responsible for expression of target genes in E. coli. Thus the pHA-M-HtrA1 plasmid can be used as a novel, rapid pre-test system for expression and cytotoxicity of the specific target gene in E. coli before assessing its functions in mammalian cells.

Quantitative Determination of High-temperature Requirement Protein A1 and Its Possible Associated Molecules during Induced Reparative Dentin Formation

IntroductionThe equilibrium of inhibitors and inducers plays an important role in the mineralization of dentin during dentinogenesis. High-temperature requirement protein A1 (HtrA1) is a novel bone mineralization inhibitor involved in physiological and pathological bone formation. However, the expression of HtrA1 in tooth mineralization is unknown. The purpose of the present study was to investigate the localization and quantity of HtrA1 and its possible related molecules during induced reparative dentin formation. MethodsRats were randomly sacrificed after direct pulp capping on days 0, 7, 14, and 21. Maxillary segments were obtained and routinely prepared for histological analysis, immunohistochemistry, quantum dots–based double immunofluorescence, and CRi’s Nuance imaging system (CRI, Woburn, MA)–based quantitative determination. ResultsThe square measure values of reparative dentin significantly increased on day 7 and continued to increase until day 21. HtrA1, matrix Gla protein (MGP), nestin, and bone sialoprotein were positively stained and colocalized in the odontoblasts and/or odontoblast-like cells zone and reparative dentin during induced reparative dentin formation. The expressions of HtrA1 and MGP were significantly enhanced after direct pulp capping on day 7 and did not significantly change between days 7, 14, and day 21. Both expressions of HtrA1 and MGP were positively correlated with the square measure values of reparative dentin; however, no correlation was found between the expressions of HtrA1 and MGP. ConclusionsHtrA1 could be observed and might possibly be involved in the process of reparative dentin formation associated with MGP.

Differential Expression of HrtA1 and ADAM12 in Placentas from Preeclamptic and Normotensive Pregnancies.

High temperature requirement factor A 1 (HtrA1) and a disintegrin and metalloproteinase 12 (ADAM12), which play roles in placental implantation and placental growth, have been implicated in the pathogenesis of preeclampsia. We investigated relative mRNA expression of both genes in placental tissues from women with preeclampsia (N=18) (average gestational age 36 weeks) and an equal number of women with normotensive pregnancies (average gestational age 39 weeks). Real-time polymerase chain reaction was used to measure mRNA extracted from term placental biopsies. Differential gene expression was evaluated using Student's T-test and fold change analyses. Statistically significant increases in placental HtRA1 (1.69-fold, p=0.030) and ADAM12 (1.48-fold, p=0.010) mRNA expression were observed among preeclamptic cases as compared with normotensive controls. HtrA1 expression was correlated with maternal age (p-value <0.01) among preeclampsia cases. Increases in HtRA1 and ADAM12 placental gene expression in placentas from preeclamptic pregnancies are consistent with some earlier reports of altered serum protein concentrations in preeclamptic pregnancies. This adds to the literature suggesting that defects in placentation (e.g. involving trophoblast invasion) are of etiologic importance in preeclampsia.

Osteoarthritis-like changes in the heterozygous sedc mouse associated with the HtrA1–Ddr2–Mmp-13 degradative pathway: a new model of osteoarthritis

To test the hypothesis that the spondyloepiphyseal dysplasia congenita (sedc) heterozygous (sedc/+) mouse, a COL2A1 mutant, is a model for the study of osteoarthritis (OA) in the absence of dwarfism and to investigate the presence of HtrA1, Ddr2, and Mmp-13 and their possible involvement in a universal mechanism leading to OA. Whole mount skeletons of adult animals were analyzed to determine whether sedc/+ mice exhibit dwarfism. To characterize progression of osteoarthritic degeneration over time, knee and temporomandibular joints from sedc/+ and wild-type mice were analyzed histologically, and severity of articular cartilage degradation was graded using the Osteoarthritis Research Society International (OARSI) scoring system. Immunohistochemistry was used to detect changes in expression of HtrA1, Ddr2, and Mmp-13 in articular cartilage of knees. As previously reported, the sedc/+ skeleton morphology was indistinguishable from wild type, and skeletal measurements revealed no significant differences. The sedc/+ mouse did, however, show significantly higher OARSI scores in knee (9, 12 and 18 months) and temporomandibular joints at all ages examined. Histological staining showed regions of proteoglycan degradation as early as 2 months in both temporomandibular and knee joints of the mutant. Cartilage fissuring and erosion were observed to begin between 2 and 6 months in temporomandibular joints and 9 months in knee joints from sedc/+ mice. Immunohistochemistry of mutant knee articular cartilage showed increased expression of HtrA1, Ddr2, and Mmp-13 compared to wild type, which upregulation preceded fibrillation and fissuring of the articular surfaces. With regard to skeletal morphology, the sedc/+ mouse appears phenotypically normal but develops premature OA as hypothesized. We conclude that the sedc/+ mouse is a useful model for the study of OA in individuals with overtly normal skeletal structure and a predisposition for articular cartilage degeneration.

HtrA1 sensitizes ovarian cancer cells to cisplatin‐induced cytotoxicity by targeting XIAP for degradation

HtrA1, a member of serine protease family, has been previously found to be involved in resistance to chemotherapy in ovarian cancer although the underlying mechanism is not clear. Using mixture-based oriented peptide library approach, previously we identified X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis proteins family, as a potential substrate of HtrA1. The aim of our work is to investigate the link between HtrA1 and XIAP proteins and their relationships with chemoresistance in ovarian cancer. Our results showed that recombinant XIAP was degraded by purified wild-type HtrA1 but not mutant HtrA1 in vitro. Consistent with the in vitro data, coimmunoprecipitation assays showed that HtrA1 and XIAP formed a protein complex in vivo. Ectopic expression of HtrA1 led to decreased level of XIAP in OV167 and OV202 ovarian cancer cells, while knockdown of HtrA1 resulted in increased level of XIAP in SKOV3 ovarian cancer cells. Furthermore, overexpression of HtrA1 in OV202 cells promoted cell sensitivity to cisplatin-induced apoptosis that could be reversed by increased expression of XIAP. The cleavage of XIAP induced by HtrA1 was enhanced by cisplatin treatment. Taken together, our experiments have identified XIAP as a novel substrate of HtrA1 and the degradation of XIAP by HtrA1 contributes to cell response to chemotherapy, suggesting that restoring the expression of HtrA1 may be a promising treatment strategy for ovarian cancer.

Abstract 4015: Flavopiridol-induced upregulation of HtrA1 is associated with suppression of its negative transcriptional regulator WT-1 and with enhanced chemosensitivity

Abstract We previously identified serine protease HtrA1 as a down-regulated gene in epithelial ovarian cancer (EOC) that modulates cisplatin-induced cytotoxicity. However, the mechanism by which HtrA1 sensitizes cells to cisplatin-induced cytotoxicity remained unknown. We have shown previously that the pan-cyclin dependent kinase inhibitor flavopiridol (FP) sensitizes cells to cisplatin-induced cytotoxicity; we hypothesized that flavopiridol might therefore modulate the expression of pro-apoptotic proteins. Microarray data indicated that while flavopiridol treatment alone or in combination with cisplatin up-regulated HtrA1 expression by 2-3 fold (providing a means to upregulate HtrA1 expression), treatment with cisplatin alone had no effect. Interestingly, in searching for underlying mechanisms, we noted that WT-1 transcription factor binding elements that is present in HtrA1 promoter is down-regulated by flavopiridol in our microarray studies. Immunoblot analysis revealed that WT-1 is highly expressed in HtrA1-deficient OV167, OV202, PE04 and Ovcar3 cells but not in HtrA1-expressing SKOV3, TOV21G and PE01 cells, suggesting WT-1 negatively regulates HtrA1 expression in EOC. Immunoblot analysis showed FP treatment resulted in down-regulation of WT-1 with a corresponding increase in HtrA1 protein levels in OV202 cells. Additionally, stable down-regulation of WT-1 by short hairpin RNA (shRNA) in OV202 cells increased HtrA1 expression. Conversely, enhanced expression of full length WT-1 isoform in SKOV3 cells downregulated HtrA1 expression. Reporter assay showed that FP treatment upregulated HtrA1 promoter activity in OV202 cells. Reporter assay and chromatin immunoprecipitation experiments showed that WT-1 is specifically recruited to the HtrA1 promoter. Moreover, downregulation of WT-1 expression in OV202 cells increased cisplatin-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays – an effect reversed by enhanced expression of HtrA1. Collectively, our studies demonstrate a linkage between the effects of flavopiridol on HtrA1 and its regulatory transcription factor WT-1 that appears in part to explain why flavopiridol has chemosensitizing properties Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4015. doi:10.1158/1538-7445.AM2011-4015

HtrA1, a potential predictor of response to cisplatin-based combination chemotherapy in gastric cancer

HtrA1 is a member of the HtrA (high-temperature requirement factor A) family of serine proteases. HtrA1 plays a protective role in various malignancies due to its tumour suppressive properties. The aim of this study was to determine HtrA1 expression as a predictor of chemoresponse in patients with advanced gastric cancer. HtrA1 expression was determined by immunohistochemistry on specimens of primary gastric cancer from 80 patients treated consecutively with cisplatin-based combination chemotherapy. Response to chemotherapy was assessed according to Response Evaluation Criteria in Solid Tumours (RECIST) criteria. Our population consisted of males/females [51/29; median age 64 years (range 32-82)]. A complete or partial response was observed in 71.4% [95% confidence interval (CI) 54.7-88.2], 66.7% (95% CI 47.8-85.5) and 28.6% (95 CI 11.8-45.3) of tumours showing high, medium and low HtrA1 expression, respectively. A statistically significant association between HtrA1 expression and the clinical response was observed (P = 0.002). The median overall survival for patients with high/medium expression was 17 months compared to 9.5 months for patients with low HtrA1 expression (P = 0.037). Identification of HtrA1 in gastric cancer prior to chemotherapy indicates that levels of HtrA1 could be used to predict response to platinum-based combination therapies. Further assessment of HtrA1 expression is highly warranted in large, prospective studies.

Expression and Functional Significance of HtrA1 Loss in Endometrial Cancer

The purpose of this study was to determine if loss of serine protease HtrA1 in endometrial cancer will promote the invasive potential of EC cell lines. Western blot analysis and immunohistochemistry methods were used to determine HtrA1 expression in EC cell lines and primary tumors, respectively. Migration, invasion assays and in vivo xenograft experiment were performed to compare the extent of metastasis between HtrA1 expressing and HtrA1 knocked down clones. Western blot analysis of HtrA1 in 13 EC cell lines revealed complete loss of HtrA1 expression in all seven papillary serous EC cell lines. Downregulation of HtrA1 in Hec1A and Hec1B cell lines resulted in a three- to fourfold increase in the invasive potential. Exogenous expression of HtrA1 in Ark1 and Ark2 cells resulted in three- to fourfold decrease in both invasive and migration potential of these cells. There was an increased rate of metastasis to the lungs associated with HtrA1 downregulation in Hec1B cells compared to control cells with endogenous HtrA1 expression. Enhanced expression of HtrA1 in Ark2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or protease deficient (SA mutant) Ark2 cells. Immunohistochemical analysis showed 57% (105/184) of primary EC tumors had low HtrA1 expression. The association of low HtrA1 expression with high-grade endometrioid tumors was statistically significant (P = 0.016). Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors.

Placental HtrA3 Is Regulated by Oxygen Tension and Serum Levels Are Altered during Early Pregnancy in Women Destined to Develop Preeclampsia

The pathogenic origin of preeclampsia is defective placental development (placentation) and function. Preeclampsia is not diagnosed until later in pregnancy, and reliable early detection is highly desirable. HtrA3 is a recently cloned gene with high expression during placentation in the mouse, rhesus monkey, and human. The present study examined the placental production and the serum profile of HtrA3 across gestation in women, the potential molecular mechanisms regulating HtrA3 production, and the association between maternal HtrA3 serum levels and preeclampsia. Immunohistochemistry determined HtrA3 expression pattern and cellular localization in first-, second-, and third-trimester placenta. The maternal serum HtrA3 levels were analyzed by Western blotting. The regulation of placental HtrA3 production and the secretion by oxygen tension was investigated in first-trimester placental explants and trophoblast cells. Placental HtrA3 protein was maximally produced in the first trimester and then dramatically down-regulated, especially in the syncytiotrophoblast. HtrA3 was secreted into the maternal circulation with a serum profile reflecting placental production. Oxygen tension regulated HtrA3; low oxygen enhanced, whereas the transition from low to high oxygen decreased, HtrA3 protein production in syncytiotrophoblast. Maternal serum HtrA3 levels at approximately 13-14 wk of gestation were significantly higher in women who subsequently developed preeclampsia. It appeared that HtrA3 down-regulation was delayed in preeclamptic pregnancies. HtrA3 protein production is closely associated with changing in oxygen tension in the placenta. The decline in HtrA3 at the end of first trimester may reflect the placental low to high oxygen switch. Abnormally high levels of serum HtrA3 at approximately 13-14 wk of gestation is associated with preeclampsia.

Genotype at Polymorphism rs11200638 and HTRA1 Expression Level

Genotype at Polymorphism rs11200638 and HTRA1 Expression Level

Inhibition of HTRA3 stimulates trophoblast invasion during human placental development

Controlled invasion of extravillous trophoblast (EVT) is necessary for implantation and placentation. The serine protease HTRA3 is highly expressed in decidual cells in the late secretory phase of the menstrual cycle and throughout pregnancy. During the first trimester it is expressed in most trophoblast cell types, but not in the invading interstitial trophoblast. HTRA3 and its family members are down-regulated in a number of cancers and are proposed as tumour-suppressors. The current study investigated whether inhibiting HTRA3 in a first trimester trophoblast cell line expressing high levels of HTRA3 would alter invasion. HTR-8/SVneo (HTR-8, derived from first trimester placenta) and a number of choriocarcinoma cells (JEG-3, AC-1M88 and AC-1M32) were screened for HTRA3 expression. Only HTR-8 cells expressed high levels of HTRA3 mRNA, consistent with HTRA3 being down-regulated in cancer. Western blotting and immunofluorescence confirmed HTRA3 protein expression and localisation in HTR-8 cells. HTRA3 was detected in conditioned medium of HTR-8 cells, confirming its secretory nature. For functional studies, both long and short forms of recombinant human HTRA3, wild type and protease-inactive mutant (S 305A) were produced using wheat-germ cell-free technology. Both have a similar molecular size, but the mutants have negligible protease activity. In addition, the mutants significantly inhibited the wild type protease activity, supporting their dominant-negative inhibition and utility as specific inhibitors of the wild type protein. Inhibition of HTRA3 by exogenous addition of HTRA3 mutant resulted in a significant increase in HTR-8 cell invasion. These results strongly support the hypothesis that HTRA3 is an inhibitor of trophoblast invasion during placental development.

The Serine Protease HtrA1 Specifically Interacts and Degrades the Tuberous Sclerosis Complex 2 Protein

Hamartin and tuberin are products of the tumor suppressor genes TSC1 and TSC2, respectively. Mutations affecting either gene result in the tuberous sclerosis syndrome, a neurologic genetic disorder characterized by the formation of multiple benign tumors or hamartomas. In this study, we report the identification of TSC2, but not TSC1, as a substrate of HtrA1, a member of the human HtrA family proteins of serine proteases. We show the direct interaction and colocalization in the cytoplasm of HtrA1 and TSC2 and that HtrA1 cleaves TSC2 both in vitro and in vivo. Finally, we show that alterations in HtrA1 expression cause modifications in phosphorylation status of two downstream targets of TSC2: 4E-BP1 and S6K. Our data suggest that, under particular physiologic or pathologic conditions, HtrA1 degrades TSC2 and activates the downstream targets. Considering that HtrA1 levels are significantly increased during embryogenesis, we speculate that one of the targets of HtrA1 activity during fetal development is the TSC2-TSC1 pathway.