An allosteric HTRA1-calpain 2 complex with restricted activation profile.

SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.

Systematic Characterization of the Clinical and Pathological Features of Schwannomas Harboring SH3PXD2A::HTRA1 Fusion.

The understanding of schwannoma tumorigenesis has been reshaped by the recent identification of SH3PXD2A::HTRA1 fusion in 10% of intracranial/spinal schwannomas. Nonetheless, pathologic features of schwannomas harboring this fusion, as well as its prevalence outside intracranial/spinal locations, have not been characterized. We screened 215 consecutive schwannomas for their clinicopathologic characteristics and fusion status using reverse-transcriptase polymerase chain reaction (RT-PCR). Among 29 (13.5%) fusion-positive schwannomas, the most prevalent location was peripheral somatic tissue (30.7%, 19/62), followed by spinal/paraspinal (18.4%, 7/38), body cavity/deep structures (10%, 2/20), intracranial (1.3%, 1/75), and viscera (0/13). All 8 cellular, 4 microcystic/reticular, and 3 epithelioid schwannomas were fusion-negative, as were 41/42 nonschwannomatous peripheral nerve sheath tumors. Remarkably, a distinct 'serpentine' palisading pattern, comprising ovoid/plump cells shorter than usual schwannian cells in a hyalinized stroma, was identified in most fusion-positive cases and the schwannomatous component of the only fusion-positive malignant peripheral nerve sheath tumor. To validate this finding, 60 additional cases were collected, including 36 with (≥10% arbitrarily) and 24 without appreciable serpentine histology, of which 29 (80.6%) and 2 (8.3%) harbored the fusion, respectively. With percentages of 'serpentine' areas scored, 10% was determined as the optimal practical cut-off to predict the fusion status (sensitivity, 0.950; specificity, 0.943). Fusion positivity was significantly associated with serpentine histology, smaller tumors, younger patients, and peripheral somatic tissue, while multivariate logistic linear regression analysis only identified serpentine histology and location as independent fusion-predicting factors. RNA in situ hybridization successfully detected the fusion junction, highly concordant with RT-PCR results. Gene expression profiling on 18 schwannomas demonstrated segregation largely consistent with fusion status. Fusion-positive cases expressed significantly higher HTRA1 mRNA abundance, perhaps exploitable as a biomarker. In summary, we systematically characterize a series of 60 SH3PXD2A::HTRA1 fusion-positive schwannomas, showing their distinctive morphology and location-specific prevalence for the first time.

HTRA1-driven detachment of type I collagen from endoplasmic reticulum contributes to myocardial fibrosis in dilated cardiomyopathy.

BACKGROUND: The aberrant secretion and excessive deposition of type I collagen (Col1) are important factors in the pathogenesis of myocardial fibrosis in dilated cardiomyopathy (DCM). However, the precise molecular mechanisms underlying the synthesis and secretion of Col1 remain unclear. METHODS AND RESULTS: RNA-sequencing analysis revealed an increased HtrA serine peptidase 1 (HTRA1) expression in patients with DCM, which is strongly correlated with myocardial fibrosis. Consistent findings were observed in both human and mouse tissues by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunofluorescence analyses. Pearson's analysis showed a markedly positive correlation between HTRA1 level and myocardial fibrosis indicators, including extracellular volume fraction (ECV), native T1, and late gadolinium enhancement (LGE), in patients with DCM. In vitro experiments showed that the suppression of HTRA1 inhibited the conversion of cardiac fibroblasts into myofibroblasts and decreased Col1 secretion. Further investigations identified the role of HTRA1 in promoting the formation of endoplasmic reticulum (ER) exit sites, which facilitated the transportation of Col1 from the ER to the Golgi apparatus, thereby increasing its secretion. Conversely, HTRA1 knockdown impeded the retention of Col1 in the ER, triggering ER stress and subsequent induction of ER autophagy to degrade misfolded Col1 and maintain ER homeostasis. In vivo experiments using adeno-associated virus-serotype 9-shHTRA1-green fluorescent protein (AAV9-shHTRA1-GFP) showed that HTRA1 knockdown effectively suppressed myocardial fibrosis and improved left ventricular function in mice with DCM. CONCLUSIONS: The findings of this study provide valuable insights regarding the treatment of DCM-associated myocardial fibrosis and highlight the therapeutic potential of targeting HTRA1-mediated collagen secretion.

Critical Dependence on Area in Relationship between ARMS2/HTRA1 Genotype and Faster Geographic Atrophy Enlargement: Age-Related Eye Disease Study 2 Report Number 33.

PURPOSE: To analyze ARMS2/HTRA1 as a risk factor for faster geographic atrophy (GA) enlargement according to (1) GA area and (2) contiguous enlargement versus progression to multifocality. DESIGN: Age-Related Eye Disease Study 2 (AREDS2) cohort analysis. PARTICIPANTS: Eyes with GA: 546 eyes of 406 participants. METHODS: Geographic atrophy area was measured from color fundus photographs at annual visits. Mixed-model regression of square root of GA area and proportional hazards regression of progression to multifocality were analyzed by ARMS2 genotype. MAIN OUTCOME MEASURES: Change in square root GA area and progression to multifocality. RESULTS: Geographic atrophy enlargement was significantly faster with ARMS2 risk alleles (P < 0.0001) at 0.224 mm/year (95% CI, 0.195-0.252 mm/year), 0.298 mm/year (95% CI, 0.271-0.324 mm/year), and 0.317 mm/year (95% CI, 0.279-0.355 mm/year), for 0 to 2 risk alleles, respectively. However, a significant interaction (P = 0.011) was observed between genotype and baseline area. In eyes with very small area (< 1.9 mm2), enlargement was significantly faster with ARMS2 risk alleles (P < 0.0001) at 0.193 mm/year (95% CI, 0.162-0.225 mm/year) versus 0.304 mm/year (95% CI, 0.280-0.329 mm/year) for 0 versus 1 to 2 risk alleles, respectively. With moderately small (1.9-3.8 mm2) or medium to large (≥ 3.8 mm2) area, enlargement was not significantly faster with ARMS2 risk alleles (P = 0.66 and P = 0.70, respectively). In nonmultifocal GA, enlargement was significantly faster with ARMS2 risk alleles (P = 0.001) at 0.175 mm/year (95% CI, 0.142-0.209 mm/year), 0.226 mm/year (95% CI, 0.193-0.259 mm/year), and 0.287 mm/year (95% CI, 0.237-0.337 mm/year) with 0 to 2 risk alleles, respectively. ARMS2 genotype was not associated significantly with progression to multifocal GA. CONCLUSIONS: The relationship between ARMS2/HTRA1 genotype and faster GA enlargement depends critically on GA area: risk alleles represent a strong risk factor for faster enlargement only in eyes with very small area. They increase the growth rate more through contiguous enlargement than progression to multifocality. ARMS2/HTRA1 genotype is more important in increasing risk of progression to GA and initial GA enlargement (contiguously) than in subsequent enlargement or progression to multifocality. These findings may explain some discrepancies between previous studies and have implications for both research and clinical practice. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Identification of highly potent and selective HTRA1 inhibitors.

High temperature requirement A serine peptidase 1 (HTRA1) is a serine protease involved in an array of signaling pathways. It is also responsible for the regulation of protein aggregates via refolding, translocation, and degradation. It has subsequently been found that runaway proteolytic HTRA1 activity plays a role in a variety of diseases, including Age-Related Macular Degeneration (AMD), osteoarthritis, and Rheumatoid Arthritis. Selective inhibition of serine protease HTRA1 therefore offers a promising new strategy for the treatment of these diseases. Herein we disclose structure-activity-relationship (SAR) studies which identify key interactions responsible for binding affinity of small molecule inhibitors to HTRA1. The study results in highly potent molecules with IC50's less than 15 nM and excellent selectivity following a screen of 35 proteases.

Decreased water exchange rate across blood-brain barrier in hereditary cerebral small vessel disease.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and heterozygous HTRA1 mutation-related cerebral small vessel disease (CSVD) are the two types of dominant hereditary CSVD. Blood-brain barrier (BBB) failure has been hypothesized in the pathophysiology of CSVD. However, it is unclear whether there is BBB damage in the two types of hereditary CSVD, especially in heterozygous HTRA1 mutation-related CSVD. In this study, a case-control design was used with two disease groups including CADASIL (n = 24), heterozygous HTRA1 mutation-related CSVD (n = 9) and healthy controls (n = 24). All participants underwent clinical cognitive assessments and brain MRI. Diffusion-prepared pseudo-continuous arterial spin labelling was used to estimate the water exchange rate across the BBB (kw). Correlation and multiple linear regression analyses were used to examine the association between kw and disease burden and neuropsychological performance, respectively. Compared with the healthy controls, kw in the whole brain and multiple brain regions was decreased in both CADASIL and heterozygous HTRA1 mutation-related CSVD patients (Bonferroni-corrected P < 0.007). In the CADASIL group, decreased kw in the whole brain (ß = -0.634, P = 0.001), normal-appearing white matter (ß = -0.599, P = 0.002) and temporal lobe (ß = -0.654, P = 0.001) was significantly associated with higher CSVD score after adjusting for age and sex. Reduced kw in the whole brain was significantly associated with poorer neuropsychological performance after adjusting for age, sex and education in both CADASIL and heterozygous HTRA1 mutation-related CSVD groups (ß = 0.458, P = 0.001; ß = 0.884, P = 0.008). This study showed that there was decreased water exchange rate across the BBB in both CADASIL and heterozygous HTRA1 mutation-related CSVD patients, suggesting a common pathophysiological mechanism underlying the two types of hereditary CSVD. These results highlight the potential use of kw for monitoring the course of CADASIL and heterozygous HTRA1 mutation-related CSVD, a possibility which should be tested in future research.

Chromosome 10q26-driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium.

Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151-171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389-407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227-3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE-Bruch's membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE-BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD.

Heterogeneous blood-brain barrier dysfunction in cerebral small vessel diseases.

INTRODUCTION: We explored how blood-brain barrier (BBB) leakage rate of gadolinium chelates (Ktrans) and BBB water exchange rate (kw) varied in cerebral small vessel disease (cSVD) subtypes. METHODS: Thirty sporadic cSVD, 40 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and 13 high-temperature requirement factor A serine peptidase 1 (HTRA) -related cSVD subjects were investigated parallel to 40 healthy individuals. Subjects underwent clinical, cognitive, and MRI assessment. RESULTS: In CADASIL, no difference in Ktrans, but lower kw was observed in multiple brain regions. In sporadic cSVD, no difference in kw, but higher Ktrans was found in the whole brain and normal-appearing white matter. In HTRA1-related cSVD, both higher Ktrans in the whole brain and lower kw in multiple brain regions were observed. In each patient group, the altered BBB measures were correlated with lesion burden or clinical severity. DISCUSSION: In cSVD subtypes, distinct alterations of kw and Ktrans were observed. The combination of Ktrans and kw can depict the heterogeneous BBB dysfunction. HIGHLIGHTS: We measured BBB leakage to gadolinium-based contrast agent (Ktrans) and water exchange rate (kw) across BBB in three subtypes of cSVD. CADASIL is characterized by lower kw, HTRA1-related cSVD exhibits both higher Ktrans and lower kw, while sporadic cSVD is distinguished by higher Ktrans. There are distinct alterations in kw and Ktrans among subtypes of cSVD, indicating the heterogeneous nature of BBB dysfunction.

HTRA1 in Placental Cell Models: A Possible Role in Preeclampsia.

The HtrA serine peptidase 1 (HTRA1) is a multidomain secretory protein with serine-protease activity involved in the regulation of many cellular processes in both physiological and pathological conditions. HTRA1 is normally expressed in the human placenta, and its expression is higher in the first trimester compared to the third trimester, suggesting an important role of this serine protease in the early phases of human placenta development. The aim of this study was to evaluate the functional role of HTRA1 in in vitro models of human placenta in order to define the role of this serine protease in preeclampsia (PE). BeWo and HTR8/SVneo cells expressing HTRA1 were used as syncytiotrophoblast and cytotrophoblast models, respectively. Oxidative stress was induced by treating BeWo and HTR8/SVneo cells with H2O2 to mimic PE conditions in order to evaluate its effect on HTRA1 expression. In addition, HTRA1 overexpression and silencing experiments were performed to evaluate the effects on syncytialization, cell mobility, and invasion processes. Our main data showed that oxidative stress significantly increased HTRA1 expression in both BeWo and HTR8/SVneo cells. In addition, we demonstrated that HTRA1 has a pivotal role in cell motility and invasion processes. In particular, HTRA1 overexpression increased while HTRA1 silencing decreased cell motility and invasion in HTR8/SVneo cell model. In conclusion, our results suggest an important role of HTRA1 in regulating extravillous cytotrophoblast invasion and motility during the early stage of placentation in the first trimester of gestation, suggesting a key role of this serine protease in PE onset.

Inhibiting HIF-1 signaling alleviates HTRA1-induced RPE senescence in retinal degeneration.

BACKGROUND: Age-related macular degeneration (AMD), characterized by the degeneration of retinal pigment epithelium (RPE) and photoreceptors, is the leading cause of irreversible vision impairment among the elderly. RPE senescence is an important contributor to AMD and has become a potential target for AMD therapy. HTRA1 is one of the most significant susceptibility genes in AMD, however, the correlation between HTRA1 and RPE senescence hasn't been investigated in the pathogenesis of AMD. METHODS: Western blotting and immunohistochemistry were used to detect HTRA1 expression in WT and transgenic mice overexpressing human HTRA1 (hHTRA1-Tg mice). RT-qPCR was used to detect the SASP in hHTRA1-Tg mice and ARPE-19 cells infected with HTRA1. TEM, SA-ß-gal was used to detect the mitochondria and senescence in RPE. Retinal degeneration of mice was investigated by fundus photography, FFA, SD-OCT and ERG. The RNA-Seq dataset of ARPE-19 cells treated with adv-HTRA1 versus adv-NC were analyzed. Mitochondrial respiration and glycolytic capacity in ARPE-19 cells were measured using OCR and ECAR. Hypoxia of ARPE-19 cells was detected using EF5 Hypoxia Detection Kit. KC7F2 was used to reduce the HIF1α expression both in vitro and in vivo. RESULTS: In our study, we found that RPE senescence was facilitated in hHTRA1-Tg mice. And hHTRA1-Tg mice became more susceptible to NaIO3 in the development of oxidative stress-induced retinal degeneration. Similarly, overexpression of HTRA1 in ARPE-19 cells accelerated cellular senescence. Our RNA-seq revealed an overlap between HTRA1-induced differentially expressed genes associated with aging and those involved in mitochondrial function and hypoxia response in ARPE-19 cells. HTRA1 overexpression in ARPE-19 cells impaired mitochondrial function and augmented glycolytic capacity. Importantly, upregulation of HTRA1 remarkably activated HIF-1 signaling, shown as promoting HIF1α expression which mainly located in the nucleus. HIF1α translation inhibitor KC7F2 significantly prevented HTRA1-induced cellular senescence in ARPE-19 cells, as well as improved the visual function in hHTRA1-Tg mice treated with NaIO3. CONCLUSIONS: Our study showed elevated HTRA1 contributes to the pathogenesis of AMD by promoting cellular senescence in RPE through damaging mitochondrial function and activating HIF-1 signaling. It also pointed out that inhibition of HIF-1 signaling might serve as a potential therapeutic strategy for AMD. Video Abstract.

Clinical features and pathogenicity assessment in patients with HTRA1-autosomal dominant disease.

BACKGROUND: Heterozygous mutations in HTRA1 were recently found to cause autosomal dominant cerebral small vessel disease (CSVD), and it was named HTRA1-autosomal dominant disease (AD-HTRA1) in the consensus recommendations of the European Academy of Neurology. This study aimed to investigate the clinical features of a mutation in HTRA1 and the effect of HTRA1 mutation on white matter hyperintensity (WMH). METHODS: A proband's brain magnetic resonance imaging (MRI) showed multiple lacunar infarctions and multiple WMH in the lateral ventricle, external capsule, frontal lobe and corpus callosum. The proband and family members were tested for CSVD-related genes by next-generation sequencing and the clinical data of the patients were collected. The published literature on AD-HTRA1 was collected, and the clinical characteristics and pathogenicity of the patients were summarized. Combined Annotation Dependent Depletion (CADD) is a tool for scoring the deleteriousness of single-nucleotide variants and insertion/deletion variants in the human genome. The relationship between the degree of WMH and the pathogenicity of the mutation was further analyzed. RESULT: It was found that the proband and her family members had a heterozygous missense mutation of c.854C > T (p.P285L) in the 4 exon of HTRA1 gene. A retrospective analysis of 5 families with c.854C > T mutation found that the patients had an early age of onset, cognitive impairment was more common, and alopecia and spondylosis could be combined at the same time. By univariate analysis, the severity of WMH was found to be significantly associated with the mutated CADD score (p < 0.05, Spearman's rho = 0.266). CONCLUSION: The clinical manifestations of AD-HTRA1 with mutation site c.854C > T (p.P285L) are similar to CARASIL, and brain MRI are mainly moderate or severe WMH and lacunar infarction (LI). WMH are affected by mutation sites. Therefore, our pathogenicity score for mutations can predict the severity of WMH.

Inorganic arsenic exposure promotes malignant progression by HDAC6-mediated down-regulation of HTRA1.

Inorganic arsenic (iAs) has been a human health concern and is associated with intestinal malignancies. However, the molecular mechanisms of the iAs-induced oncogenic process in intestine epithelial cells remain elusive, partly because of the known hormesis effect of arsenic. Here, we established that six-month exposure to iAs at a concentration similar to those found in contaminated drinking water could promote malignant characteristics, including enhanced proliferation and migration, resistance to apoptosis, and mesenchymal-like transition in Caco-2 cells. Transcriptome analysis and mechanism study revealed that key genes and pathways involved in cell adhesion, inflammation and oncogenic regulation were altered during chronic iAs exposure. Specifically, we uncovered that down-regulation of HTRA1 was essential for the iAs-induced acquisition of the cancer hallmarks. Further, we evidenced that the loss of HTRA1 during iAs-exposure could be restored by HDAC6 inhibition. Caco-2 cells with chronic exposure to iAs exhibited enhanced sensitivity to WT-161, a specific inhibitor of HDAC6, when used alone than in combination with a chemotherapeutic agent. These findings provide valuable information for understanding the mechanisms of arsenic-induced carcinogenesis and facilitating the health management of populations in arsenic-polluted areas.

Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the global aging population. Familial aggregation and genome-wide association (GWA) studies have identified gene variants associated with AMD, implying a strong genetic contribution to AMD development. Two loci, on human Chr 1q31 and 10q26, respectively, represent the most influential of all genetic factors. While the role of CFH at Chr 1q31 is well established, uncertainty remains about the genes ARMS2 and HTRA1, at the Chr 10q26 locus. Since both genes are in strong linkage disequilibrium, assigning individual gene effects is difficult. In this chapter, we review current literature about ARMS2 and HTRA1 and their relevance to AMD risk. Future studies will be necessary to unravel the mechanisms by which they contribute to AMD.

Activation by substoichiometric inhibition.

Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed.

Rare neurovascular genetic and imaging markers across neurodegenerative diseases.

INTRODUCTION: Cerebral small vessel disease (SVD) is common in patients with cognitive impairment and neurodegenerative diseases such as Alzheimer's and Parkinson's. This study investigated the burden of magnetic resonance imaging (MRI)-based markers of SVD in patients with neurodegenerative diseases as a function of rare genetic variant carrier status. METHODS: The Ontario Neurodegenerative Disease Research Initiative study included 520 participants, recruited from 14 tertiary care centers, diagnosed with various neurodegenerative diseases and determined the carrier status of rare non-synonymous variants in five genes (ABCC6, COL4A1/COL4A2, NOTCH3/HTRA1). RESULTS: NOTCH3/HTRA1 were found to significantly influence SVD neuroimaging outcomes; however, the mechanisms by which these variants contribute to disease progression or worsen clinical correlates are not yet understood. DISCUSSION: Further studies are needed to develop genetic and imaging neurovascular markers to enhance our understanding of their potential contribution to neurodegenerative diseases.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL): A challenging diagnosis and a rare multiple sclerosis mimic.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an extremely rare hereditary cerebral small vessel disease caused by homozygous or compound heterozygous mutations in the gene coding for high-temperature requirement A serine peptidase 1 (HtrA1). Given the rare nature of the disease, delays in diagnosis and misdiagnosis are not uncommon. In this article, we reported the first case of CARASIL from Saudi Arabia with a novel homozygous variant c.1156C>T in exon 7 of the HTRA1 gene. The patient was initially misdiagnosed with primary progressive multiple sclerosis and treated with rituximab. CARASIL should be considered in the differential diagnosis of patients with suspected atypical progressive multiple sclerosis who have additional signs such as premature scalp alopecia and low back pain with diffuse white matter lesions in brain MRI. Genetic testing is important to confirm the diagnosis.

Lipopolysaccharide Activating NF-kB Signaling by Regulates HTRA1 Expression in Human Retinal Pigment Epithelial Cells.

Inflammation and elevated expression of high temperature requirement A serine peptidase 1 (HTRA1) are known high risk factors for age-related macular degeneration (AMD). However, the specific mechanism that HTRA1 causes AMD and the relationship between HTRA1 and inflammation remains unclear. We found that lipopolysaccharide (LPS) induced inflammation enhanced the expression of HTRA1, NF-κB, and p-p65 in ARPE-19 cells. Overexpression of HTRA1 up-regulated NF-κB expression, and on the other hand knockdown of HTRA1 down-regulated the expression of NF-κB. Moreover, NF-κB siRNA has no significant effect on the expression of HTRA1, suggesting HTRA1 works upstream of NF-κB. These results demonstrated that HTRA1 plays a pivotal role in inflammation, explaining possible mechanism of overexpressed HTRA1-induced AMD. Celastrol, a very common anti-inflammatory and antioxidant drug, was found to suppress inflammation by inhibiting phosphorylation of p65 protein efficaciously in RPE cells, which may be applied to the therapy of age-related macular degeneration.

Binding of Gtf2i-ß/δ transcription factors to the ARMS2 gene leads to increased circulating HTRA1 in AMD patients and in vitro.

The disease-initiating molecular events for age-related macular degeneration (AMD), a multifactorial retinal disease affecting many millions of elderly individuals worldwide, are still unknown. Of the over 30 risk and protective loci so far associated with AMD through whole genome-wide association studies (GWAS), the Age-Related Maculopathy Susceptibility 2 (ARMS2) gene locus represents one of the most highly associated risk regions for AMD. A unique insertion/deletion (in/del) sequence located immediately upstream of the High Temperature Requirement A1 (HTRA1) gene in this region confers high risk for AMD. Using electrophoretic mobility shift assay (EMSA), we identified that two Gtf2i-ß/δ transcription factor isoforms bind to the cis-element 5'- ATTAATAACC-3' contained in this in/del sequence. The binding of these transcription factors leads to enhanced upregulation of transcription of the secretory serine protease HTRA1 in transfected cells and AMD patient-derived induced pluripotent stem cells (iPSCs). Overexpression of Htra1 in mice using a CAG-promoter demonstrated increased blood concentration of Htra1 protein, caused upregulation of vascular endothelial growth factor (VEGF), and produced a choroidal neovascularization (CNV)-like phenotype. Finally, a comparison of 478 AMD patients to 481 healthy, age-matched controls from Japan, India, Australia, and the USA showed a statistically increased level of secreted HTRA1 blood concentration in AMD patients compared with age-matched controls. Taken together, these results suggest a common mechanism across ethnicities whereby increased systemic blood circulation of secreted serine protease HTRA1 leads to subsequent degradation of Bruch's membrane and eventual CNV in AMD.

Forward and reverse degradomics defines the proteolytic landscape of human knee osteoarthritic cartilage and the role of the serine protease HtrA1.

OBJECTIVES: Proteolytic destruction of articular cartilage, a major pathogenic mechanism in osteoarthritis (OA), was not previously investigated by terminomics strategies. We defined the degradome of human knee OA cartilage and the contribution therein of the protease HtrA1 using Terminal Amine Isotopic Labeling of Substrates (TAILS). DESIGN: Proteins from OA cartilage taken at knee arthroplasty (n = 6) or separately, from healthy cartilage incubated in triplicate with/without active HtrA1, were labeled at natural and proteolytically cleaved N-termini by reductive dimethylation, followed by trypsin digestion, enrichment of N-terminally labeled/blocked peptides, tandem mass spectrometry and positional peptide annotation to identify cleavage sites. Biglycan proteolysis by HtrA1 was validated biochemically and Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) was used to define the HtrA1 cleavage sites. RESULTS: We identified 10,155 unique internal peptides from 2,162 proteins, suggesting at least 10,797 cleavage sites in OA cartilage. 7,635 internal peptides originated in 371 extracellular matrix/secreted components, many undergoing extensive proteolysis. Rampant ragging of protein termini suggested pervasive exopeptidase activity. HtrA1, the most abundant protease in OA cartilage, experimentally generated 323 cleavages in 109 cartilage proteins, accounting for 171 observed cleavages in the OA degradome. ATOMS identified HtrA1 cleavage sites in a selected substrate, biglycan, whose direct cleavage by HtrA1 was thus orthogonally validated. CONCLUSIONS: OA cartilage demonstrates widespread proteolysis by endo- and exopeptidases. HtrA1 contributes broadly to cartilage proteolysis. Forward degradomics of OA cartilage together with reverse degradomics of proteases active in OA, e.g., HtrA1, can potentially fully annotate OA proteolytic pathways and provide new biomarkers.

The Phenotypic Course of Age-Related Macular Degeneration for ARMS2/HTRA1: The EYE-RISK Consortium.

PURPOSE: Age-related maculopathy susceptibility 2 (ARMS2) is considered the most enigmatic of the genes for age-related macular degeneration (AMD). We investigated the phenotypic course and spectrum of AMD for the risk haplotype at the ARMS2 and high-temperature requirement A serine peptidase 1 (HTRA1) locus in a large European consortium. DESIGN: Pooled analysis of 4 case-control and 6 cohort studies. PARTICIPANTS: Individuals (N = 17 204) aged 55 years or older participating in the European Eye Epidemiology consortium. METHODS: Age-related macular degeneration features and macular thickness were determined on multimodal images; data on genetics and phenotype were harmonized. Risks of AMD features for rs3750486 genotypes at the ARMS2/HTRA1 locus were determined by logistic regression and were compared with a genetic risk score (GRS) of 19 variants at the complement pathway. Lifetime risks were estimated with Kaplan-Meier analyses in population-based cohorts. MAIN OUTCOME MEASURES: Age-related macular degeneration features and stage. RESULTS: Of 2068 individuals with late AMD, 64.7% carried the ARMS2/HTRA1 risk allele. For homozygous carriers, the odds ratio (OR) of geographic atrophy was 8.6 (95% confidence interval [CI], 6.5-11.4), of choroidal neovascularization (CNV) was 11.2 (95% CI, 9.4-13.3), and of mixed late AMD was 12.2 (95% CI, 7.3-20.6). Cumulative lifetime risk of late AMD ranged from 4.4% for carriers of the nonrisk genotype to 9.4% and 26.8% for heterozygous and homozygous carriers. The latter received the diagnosis of late AMD 9.6 years (95% CI, 8.0-11.2) earlier than carriers of the nonrisk genotype. The risk haplotype was not associated with hard or soft drusen < 125 µm (OR, 1.2; 95% CI, 0.9-1.7), but risks increased significantly for soft drusen ≥ 125 µm (OR, 2.1; 95% CI, 1.5-3.0), up to an OR of 7.2 (95% CI, 3.8-13.8) for reticular pseudodrusen. Compared with persons with a high GRS for complement, homozygous carriers of ARMS2/HTRA1 showed a higher risk of CNV (OR, 4.1; 95% CI, 3.2-5.4); risks of other characteristics were not different. CONCLUSIONS: Carriers of the risk haplotype at ARMS2/HTRA1 have a particularly high risk of late AMD at a relatively early age. Data suggest that risk variants at ARMS2/HTRA1 act as a strong catalyst of progression once early signs are present. The phenotypic spectrum resembles that of complement genes, only with higher risks of CNV.