High-temperature Requirement Protein A1 Research Articles

Carfilzomib relieves pancreatitis-initiated pancreatic ductal adenocarcinoma by inhibiting high-temperature requirement protein A1

Pancreatitis is a crucial risk factor for pancreatic ductal adenocarcinoma (PDAC), and our previous study had proved high-temperature requirement protein A1 (HTRA1) exacerbates pancreatitis insult; however, the function and mechanism of HTRA1 in pancreatitis-initiated PDAC is still unclear. In the present paper, we clarified the expression of HTRA1 in PDAC using bioinformatics and immunohistochemistry of tissue chip, and found that HTRA1 is significantly upregulated in PDAC. Moreover, the proliferation, migration, invasion and adhesion of PANC-1 and SW1990 cells were promoted by overexpression of HTRA1, but inhibited by knockdown of HTRA1. Meanwhile, we found that HTRA1 arrested PANC-1 and SW1990 cells at G2/M phase. Mechanistically, HTRA1 interacted with CDK1 protein, and CDK1 inhibitor reversed the malignant phenotype of PANC-1 and pancreatitis-initiated PDAC activated by HTRA1 overexpression. Finally, we discovered a small molecule drug that can inhibit HTRA1, carfilzomib, which has been proven to inhibit the biological functions of tumor cells in vitro and intercept the progression of pancreatitis-initiated PDAC in vivo. In conclusion, the activation of HTRA1-CDK1 pathway promotes the malignant phenotype of tumor cells by blocking the cell cycle at the G2/M phase, thereby accelerating pancreatitis-initiated PDAC. Carfilzomib is an innovative candidate drug that can inhibit pancreatitis-initiated PDAC through targeted inhibition of HTRA1.

Long non-coding RNA lincRNA-erythroid prosurvival (EPS) alleviates cerebral ischemia/reperfusion injury by maintaining high-temperature requirement protein A1 (Htra1) stability through recruiting heterogeneous nuclear ribonucleoprotein L (HNRNPL)

ABSTRACT This study aimed at investigating the role and mechanism of lincRNA-EPS (erythroid prosurvival) in cerebral ischemia/reperfusion (CIR) injury. The results showed that the overexpression of lincRNA-EPS was able to reduce the levels of interleukin-6, tumor necrosis factor-alpha and interleukin-1β stimulated in the OGD-treated Neuro-2a (N-2a) cells. The levels of reactive oxygen species and malondialdehyde were enhanced while the superoxide dismutase levels were reduced by oxygen and glucose deprivation (OGD) treatment, in which the lincRNA-EPS overexpression could reverse this effect in the cells. LincRNA-EPS interacted with high-temperature requirement protein A1 (Htra1) and heterogeneous nuclear ribonucleoprotein L (HNRNPL), and their depletion inhibited the Htra1 mRNA stability in N-2a cells. HNRNPL knockdown blocked lincRNA-EPS overexpression-induced Htra1 expression in the cells. The depletion of Htra1 could rescue lincRNA-EPS overexpression-mediated N-2a cell injury, inflammation, and oxidative stress induced by OGD. Functionally, lincRNA-EPS alleviates CIR injury of the middle cerebral artery occlusion/reperfusion mice in vivo. In conclusion, lincRNA-EPS attenuates CIR injury by maintaining Htra1 stability through recruiting HNRNPL.

Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates

Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-β (Aβ) peptides and formation of Aβ deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aβ pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC–MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aβ1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aβ deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.

Phase 1 Study of the Anti-HtrA1 Antibody-binding Fragment FHTR2163 in Geographic Atrophy Secondary to Age-related Macular Degeneration

FHTR2163 is a novel antigen-binding fragment (Fab) directed against high-temperature requirement protein A1 (HtrA1). HTRA1 inhibition may preserve retinal integrity and slow disease progression in geographic atrophy (GA) secondary to age-related macular degeneration (AMD). This study examined the safety, pharmacokinetics, immunogenicity, and changes in the HTRA1-specific substrate Dickkop-related protein 3 (DKK3) in patients with GA who received FHTR2163. Phase I, open-label, single ascending dose escalation and multiple-dose expansion study. Adults aged ≥ 50 years with GA secondary to AMD with best corrected visual acuity ranging between Snellen 20/125 and 20/400 were enrolled. In the first stage, a single intravitreal injection of FHTR2163 was given in 5 dose-escalation cohorts ranging from 1 to 20 mg (n=3 patients/cohort; n=15 total patients). The second stage evaluated the maximum tested dose of 20mg administered every 4 weeks for 3 doses (n=13 patients). No dose limiting toxicities or ocular serious AEs were reported. The most frequently reported AEs in the study eye were conjunctival hemorrhage (n=7), conjunctival hyperemia (n=4), and eye pain (n=2). No non-ocular or ocular AEs were assessed as drug related. There were no clinically significant changes in ocular exams. A sustained pharmacodynamic effect of anti-HtrA1 was observed in the aqueous humor, as measured by levels of cleaved DKK3. FHTR2163, a novel Fab directed against HtrA1, was well tolerated with no DLTs or significant ocular AEs. The molecule when injected intravitreally for 3 doses showed a sustained pharmacodynamic effect at the maximum tested dose of 20 mg.

HTRA1 Variants and the Interaction with Smoking Confer the Genetic Susceptibility to Ischemic Stroke.

High temperature requirement protein A1 (HtrA1) was identified as the causative gene of autosomal recessive arteriopathy and associated with lacunar ischemic stroke (IS) in European. This study aimed at evaluating the association of HTRA1 with IS and four tagging single-nucleotide polymorphisms (SNPs) were genotyped in a cohort of 4,098 Chinese. The mRNA level of HTRA1 in 72 IS cases and 72 hypertension controls were measured and compared. In whole population, SNP rs2268350 (C>T) was significantly associated with IS incidence (P=0.034). Stratification analysis observed significant association of rs2268350 in male, smoking and drinking populations, rs2672587 (C>G) in smoking and nonsmoking populations and rs3793917 (C>G) in smoking, nonsmoking and nondrinking populations with stroke respectively (P<0.05). The additive interaction and multiplicative interaction between rs2268350 and smoking were both of significant (P<0.05) after adjustment for the covariates. There was a cumulated risk of IS among genotypes of rs3793917 (P=0.009) and rs2672587 (P=0.047) in smoking population. The mRNA level of HTRA1 in non-smokers with rs2268350 CC was significantly higher than smokers with rs2268350 CT/TT (P=0.046) in IS cases. Our findings support that HTRA1 confers the genetic susceptibility to IS and smoking might modify the genetic effect of HTRA1 on IS by suppressing HTRA1 mRNA expression.

Tetrandrine partially reverses multidrug resistance of human laryngeal cancer cells.

ObjectiveStudies have demonstrated that tetrandrine reverses multidrug resistance (MDR) in animal models or cell lines derived from multiple cancer types. We examined the potential MDR reversal activity of tetrandrine in a multidrug-resistant variant of a human laryngeal cancer Hep-2 cell line and explored potential mechanisms involved.MethodsWe developed the multidrug-resistant variant cell line (Hep-2/v) by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (VCR). After Hep-2 or Hep-2/v cells were treated with tetrandrine (2.52 µg/mL), MDR was measured by MTT assay, rhodamine 123 retention was measured by flow cytometry, and mRNA and protein expression of multidrug resistance 1 (MDR1), regulator of G-protein signaling 10 (RGS10), high-temperature requirement protein A1 (HTRA1), and nuclear protein 1 (NUPR1) were detected by real-time reverse transcription-PCR and western blotting, respectively.ResultsTetrandrine significantly lowered the half-maximal inhibitory concentration (IC50) of VCR in Hep-2/v cells, resulting in a 2.22-fold reversal of MDR. Treatment with tetrandrine increased rhodamine 123 retention, downregulated the mRNA and protein expression of MDR1 and RGS10, and upregulated expression of HTRA1 in Hep-2/v cells.ConclusionWe showed that tetrandrine exerts anti-MDR activity in Hep-2/v cells, possibly by inhibiting MDR1 overexpression-mediated drug efflux and by altering expression of HTRA1 and RGS10.

The serine protease HtrA1 cleaves misfolded transforming growth factor β–induced protein (TGFBIp) and induces amyloid formation

The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor β–induced protein (TGFBIp)–linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2DE analyses, and thioflavin T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave WT TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the WT FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming in vitro amyloid aggregates. Significantly, these peptides have been previously identified in amyloid deposits in vivo, supporting the idea that HtrA1 is a causative agent for TGFBIp-associated amyloidosis in corneal dystrophy. In summary, our results indicate that TGFBIp is an HtrA1 substrate and that some mutations in the gene encoding TGFBIp cause aberrant HtrA1-mediated processing that results in amyloidogenesis in corneal dystrophies.

CADASIL brain vessels show a HTRA1 loss-of-function profile.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and a phenotypically similar recessive condition (CARASIL) have emerged as important genetic model diseases for studying the molecular pathomechanisms of cerebral small vessel disease (SVD). CADASIL, the most frequent and intensely explored monogenic SVD, is characterized by a severe pathology in the cerebral vasculature including the mutation-induced aggregation of the Notch3 extracellular domain (Notch3ECD) and the formation of protein deposits of insufficiently determined composition in vessel walls. To identify key molecules and pathways involved in this process, we quantitatively determined the brain vessel proteome from CADASIL patient and control autopsy samples (n = 6 for each group), obtaining 95 proteins with significantly increased abundance. Intriguingly, high-temperature requirement protein A1 (HTRA1), the extracellular protease mutated in CARASIL, was found to be strongly enriched (4.9-fold, p = 1.6 × 10-3) and to colocalize with Notch3ECD deposits in patient vessels suggesting a sequestration process. Furthermore, the presence of increased levels of several HTRA1 substrates in the CADASIL proteome was compatible with their reduced degradation as consequence of a loss of HTRA1 activity. Indeed, a comparison with the brain vessel proteome of HTRA1 knockout mice (n = 5) revealed a highly significant overlap of 18 enriched proteins (p = 2.2 × 10-16), primarily representing secreted and extracellular matrix factors. Several of them were shown to be processed by HTRA1 in an invitro proteolysis assay identifying them as novel substrates. Our study provides evidence for a loss of HTRA1 function as a critical step in the development of CADASIL pathology linking the molecular mechanisms of two distinct SVD forms.

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.

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.

HtrA1 may regulate the osteogenic differentiation of human periodontal ligament cells by TGF-β1.

Periodontal ligament cells (PDLCs) in periodontal ligament (PDL) can differentiate into osteoblasts, while physiologically PDL remains non-mineralized space although located two hard tissues. But the exact mechanism of which is still unclear. High-temperature requirement protein A1 (HtrA1) is a key mineralization regulator and could inhibit the osteogenesis by transforming growth factor-β (TGF-β) signaling. However, the role of HtrA1 in PDLCs osteogenic differentiation has yet to be clarified. We assume HtrA1 may play an important role in maintaining the balance of PDL mineralization, and may regulate human periodontal ligament cells (hPDLCs) osteogenic differentiation by TGF-β1. Firstly we confirmed the mRNA expression of HtrA1 and TGF-β1 in hPDLCs by RT-PCR, then QDs-based immunofluorescence demonstrated the co-localization of them in the cytoplasm, and co-immunoprecipitation further confirmed the interaction between them. Lentivirus-mediated HtrA1 overexpression enhanced the osteogenic differentiation of hPDLCs, as well as up-regulation of TGF-β1. In contrast, knockdown of HtrA1 suppressed the osteogenic differentiation with down-regulation of TGF-β1. These findings suggested that HtrA1 plays a positive role in hPDLCs osteogenic differentiation and may regulate this process by TGF-β1.

Protoporphyrins enhance oligomerization and enzymatic activity of HtrA1 serine protease.

High temperature requirement protein A1 (HtrA1), a secreted serine protease of the HtrA family, is associated with a multitude of human diseases. However, the exact functions of HtrA1 in these diseases remain poorly understood. We seek to unravel the mechanisms of HtrA1 by elucidating its interactions with chemical or biological modulators. To this end, we screened a small molecule library of 500 bioactive compounds to identify those that alter the formation of extracellular HtrA1 complexes in the cell culture medium. An initial characterization of two novel hits from this screen showed that protoporphyrin IX (PPP-IX), a precursor in the heme biosynthetic pathway, and its metalloporphyrin (MPP) derivatives fostered the oligomerization of HtrA1 by binding to the protease domain. As a result of the interaction with MPPs, the proteolytic activity of HtrA1 against Fibulin-5, a specific HtrA1 substrate in age-related macular degeneration (AMD), was increased. This physical interaction could be abolished by the missense mutations of HtrA1 found in patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Furthermore, knockdown of HtrA1 attenuated apoptosis induced by PPP-IX. These results suggest that PPP-IX, or its derivatives, and HtrA1 may function as co-factors whereby porphyrins enhance oligomerization and the protease activity of HtrA1, while active HtrA1 elevates the pro-apoptotic actions of porphyrin derivatives. Further analysis of this interplay may shed insights into the pathogenesis of diseases such as AMD, CARASIL and protoporphyria, as well as effective therapeutic development.

The Autolysis of Human HtrA1 Is Governed by the Redox State of Its N-Terminal Domain

Human HtrA1 (high-temperature requirement protein A1) belongs to a conserved family of serine proteases involved in protein quality control and cell fate. The homotrimeric ubiquitously expressed protease has chymotrypsin-like specificity and primarily targets hydrophobic stretches in selected or misfolded substrate proteins. In addition, the enzyme is capable of exerting autolytic activity by removing the N-terminal insulin-like growth factor binding protein (IGFBP)/Kazal-like tandem motif without affecting the protease activity. In this study, we have addressed the mechanism governing the autolytic activity and find that it depends on the integrity of the disulfide bonds in the N-terminal IGFBP/Kazal-like domain. The specificity of the autolytic cleavage reveals a strong preference for cysteine in the P1 position of HtrA1, explaining the lack of autolysis prior to disulfide reduction. Significantly, the disulfides were reduced by thioredoxin, suggesting that autolysis of HtrA1 in vivo is linked to the endogenous redox balance and that the N-terminal domain acts as a redox-sensing switch.

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

Elevated serine protease HtrA1 inhibits cell proliferation, reduces invasion, and induces apoptosis in esophageal squamous cell carcinoma by blocking the nuclear factor-κB signaling pathway

Emerging evidence has demonstrated that high-temperature requirement protein A1 (HtrA1) appears to be involved in several important biological processes in mammals such as growth, apoptosis, embryogenesis, invasion, metastasis, and cancer and has been verified to be reduced in a variety of human tumors. However, its precise functions and molecular mechanisms in esophageal squamous cell carcinoma (ESCC) remain unclear. Here, we detected HtrA1 level in ESCC tissues and cells and investigated the biological roles of HtrA1 in ESCC. We found that expressions of HtrA1 mRNA and protein in ESCC tissues and cells were significantly lower than those in normal esophageal epithelial tissues and cells (P < 0.05). Expressions of HtrA1 mRNA and protein were closely associated with TNM staging and lymph node metastasis (P < 0.05). Additionally, the survival rate of patients with low HtrA1 level was lower than those patients with high HtrA1 level (P < 0.05). Elevated HtrA1 level markedly inhibited cell proliferation in vitro and in vivo, reduced cell invasion in vitro, and induced cell apoptosis. Notably, HtrA1 overexpression inhibited phosphorylation levels of IκBα and p65 subunit of the NF-κB signaling pathway, but increased total IκBα level, coupled with decreases of Ki-67, Bcl-2, Bcl-xL, cyclin D1, and MMP-9 proteins and increase of caspase-3 activity. Overall, these data suggest that HtrA1 may play critical roles in the tumorgenesis and progression of ESCC, and HtrA1 overexpression exerts its anti-tumor effect by blocking the NF-κB signaling pathway; thus, manipulation of HtrA1 may be an effective molecular target for ESCC treatment.

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.

A novel ARMS2 splice variant is identified in human retina

Age-related macular degeneration (AMD, MIM 603075) is the major cause of irreversible visual impairment in developed countries. AMD damages photoreceptors located in the central region of the retina (the macula), resulting in an impairment of central visual acuity (Jager et al., 2008). A locus at chromosome 10q26 has been consistently linked to the disease and represents one of the two strongest genetic effects discovered (Weeks et al., 2001; Majewski et al., 2003; Weeks et al., 2004; Kenealy et al., 2004; Fisher et al., 2005). However, it has been difficult to identify with certainty the genetic variant at this locus biologically responsible for modulating risk of AMD. Studies have shown that the AMD-associated haplotype includes thesingle nucleotide polymorphisms (SNPs) rs10490924 in gene ARMS2 (Age-related maculopathy susceptibility 2, previously known as hypothetical LOC387715) and rs11200638 in the promoter region of HTRA1 (High-temperature requirement protein A1) (Jakobsdottir et al., 2005; Rivera et al., 2005; Schmidt et al., 2006; Yang et al., 2006; Dewan et al., 2006; Kanda et al., 2007; Fritsche et al., 2008). Unfortunately, the variations in genes ARMS2 and HTRA1 are in such strong linkage disequilibrium (LD) that their effects are indistinguishable using statistical analysis. While studies have suggested a functional effect for each SNP, evidence that the HTRA1 polymorphism is functional and influences gene expression is inconsistent (Yang et al., 2006; Chan et al., 2007; Kanda et al., 2007; Chowers et al., 2008; Tuo et al., 2008; Kanda et al., 2010; Wang et al., 2010; Yang et al., 2010; Friedrich et al., 2011). This inconsistency, combined with the observation that the ARMS2 rs10490924 variant changes the corresponding amino acid sequence of the protein, suggests that ARMS2 is the more likely AMD gene in this region, although this continues to be debated in the literature.

747: Differential expression of high-temperature requirement protein A1 and A2 in human placentas and cord blood from normal and preeclamptic pregnancies

747: Differential expression of high-temperature requirement protein A1 and A2 in human placentas and cord blood from normal and preeclamptic pregnancies

High-Temperature Requirement Protein A1 (HtrA1) Gene Expression in Dog Mandible Bone by &lt;I&gt;&amp;beta;&lt;/I&gt;-TCP

Beta-tricalcium phosphate (β-TCP) has been used for bone regeneration in a variety of surgical procedures including dental implant therapy with satisfactory clinical results. However, very little is known about the molecular basis mechanisms enhancing the bone formation by β-TCP. To understand the molecular basis mechanism of β-TCP in bone formation, β-TCP was implanted into bone defects of mandible bones in beagle dogs. After 4, 7 and 14 days, bone tissues during the healing process were recovered and gene expression profiles were examined using an Affymetrix GeneChip system. A significantly higher high-temperature requirement protein A1 (HtrA1) mRNA level was found in the 4-day samples after β-TCP implantation compared with the controls. The elevated HtrA1 gene expressions in β-TCP-implanted bone tissues were confirmed by RT-PCR and real-time PCR. Because HtrA1 is known as a key regulator of physiological and pathological matrix mineralization, the enhancement of HtrA1 gene expression in β-TCP-implanted mandible bone might be one of the molecular mechanisms for stimulating bone formation.