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Permanent cerebral ischemia is a consequence of prolonged cerebral artery occlusion that results in severe brain damage. Neurotoxicity occurring after ischemia can induce brain tissue damage by destroying cell organelles and their function. Neferine is a natural compound isolated from the seed embryos of the lotus plant and has broad pharmacological effects, including blockading of the calcium channels, anti-oxidative stress, and anti-apoptosis. This study investigated the ability of neferine to reduce brain injury after permanent cerebral occlusion. Permanent cerebral ischemia in rats was induced by instigation of occlusion of the middle cerebral artery for 24 h. The rats were divided into 6 groups: sham, permanent middle cerebral artery occlusion (pMCAO), pMCAO with neferine and nimodipine treatment. To investigate the severity of the injury, the neurological deficit score and morphological alterations were investigated. After 24 h, the rats were evaluated to assess neurological deficit, infarct volume, morphological change, and the number of apoptotic cell deaths. In addition, the brain tissues were examined by western blot analysis to calculate the expression of proteins related to oxidative stress and apoptosis. The data showed that the neurological deficit scores and the infarct volume were significantly reduced in the neferine-treated rats compared to the vehicle group. Treatment with neferine significantly reduced oxidative stress with a measurable decrease in 4-hydroxynonenal (4-HNE), nitric oxide (NO), neuronal nitric oxide (nNOS), and calcium levels and an upregulation of Hsp70 expression. Neferine treatment also significantly decreased apoptosis, with a decrease in Bax and cleaved caspase-3 and an increase in Bcl-2. This study suggested that neferine had a neuroprotective effect on permanent cerebral ischemia in rats by diminishing oxidative stress and apoptosis.

The high temperature requirement factor A1 (HTRA1) is a serine protease which modulates an array of signalling pathways driving basal biological processes. HTRA1 plays a significant role in cell proliferation, migration and fate determination, in addition to controlling protein aggregates through refolding, translocation or degradation. The mutation of HTRA1 has been implicated in a plethora of disorders and this has also led to its growing interest as drug therapy target. This review details the involvement of HTRA1 in certain signalling pathways, namely the transforming growth factor beta (TGF-β), canonical Wingless/Integrated (WNT) and NOTCH signalling pathways during organogenesis and various disease pathogenesis such as preeclampsia, age-related macular degeneration (AMD), small vessel disease and cancer. We have also explored possible avenues of exploiting the serine proteases for therapeutic management of these disorders.

Neferine is the major alkaloid compound isolated from the seed embryos of lotus. Neferine has many pharmacological effects, such as anti-inflammatory, antioxidative stress, and antiapoptotic effects, and it maintains autophagic balance. The purpose of this study was to explore the mechanism by which neferine attenuates autophagy after permanent cerebral ischemia in rats. We performed permanent cerebral ischemia in rats by middle cerebral artery occlusion (pMCAO) for 12 h with or without administration of neferine or nimodipine, a calcium (Ca2+) channel blocker. Neuroprotective effects were determined by evaluating the infarct volume and neurological deficits. Autophagy and its signaling pathway were determined by evaluating the expression of phosphorylated AMP-activated protein kinase alpha (AMPKα), phosphorylated mammalian target of rapamycin (mTOR), beclin-1, microtubule-associated protein 1A/1B-light chain 3 class II (LC3-II), and p62 by western blotting. Autophagosomes were evaluated by transmission electron microscopy. Neferine treatment significantly reduced infarct volumes and improved neurological deficits. Neferine significantly attenuated the upregulation of autophagy-associated proteins such as LC3-II, beclin-1, and p62 as well as autophagosome formation, all of which were induced by pMCAO. Neferine exerted remarkable protection against cerebral ischemia, possibly via the regulation of autophagy mediated by the Ca2+-dependent AMPK/mTOR pathway.

Nonalcoholic fatty liver disease (NAFLD) is relatively associated with comorbidities in obesity and metabolic inflammation. Low-grade inflammation following the high-fat diet (HFD)-induced NAFLD can promote the development of nonalcoholic steatohepatitis (NASH) through particularly liver-resident immune cell recruitment and hepatic nuclear factor kappa B (NF-κB) pathway. Therefore, inflammatory intervention may contribute to NASH reduction. Pelargonic acid vanillylamide (PAVA) or nonivamide is one of the pungent capsaicinoids of Capsicum species and has been found in chili peppers. Our previous study demonstrated that PAVA improved hepatic function, decreased oxidative stress and reduced apoptotic cell death but the insight role of PAVA on NAFLD is still unclear. Thus, this study aimed to investigate the underlying anti-inflammatory mechanism of PAVA in an NAFLD-rat model. Male Sprague Dawley rats were fed with normal diet or HFD for 16 weeks. Then high-fat rats were given vehicle or PAVA (1 mg/kg/day) for another 4 weeks. We found that PAVA alleviated hepatic inflammation associated with the reducing toll-like receptor 4/NF-κB pathway, showing significantly lower recruitment of cluster of differentiation 44. PAVA also maintained activity of insulin signaling pathway, and attenuated NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome formation. NAFLD progresses to NASH through transforming growth factor (TGF-β1), and also recovery to simple stage followed by PAVA suppresses pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, interleukin-6, and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. Therefore, our findings suggest that PAVA provides a novel therapeutic approach for NAFLD and slows the progression to NASH.

Objectives: Mammalian DNA methyltransferases are essential to re-establish global DNA methylation patterns during implantation, which is critical for transmitting epigenetic information to the next generation. In contrast, the significance of methyl-CpG binding proteins (MBPs) that bind methylated CpG remains almost unknown at this stage. We previously demonstrated that Zbtb38 (also known as CIBZ)—a zinc finger type of MBP—is required for mouse embryonic stem (ES) cell proliferation by positively regulating Nanog expression. However, the physiological function of Zbtb38 in vivo remains unclear. Materials and Methods: This study used the Cre-loxP system to generate conditional Zbtb38 knockout mice. Cell proliferation and apoptosis were studied by immunofluorescence staining. Quantitative real-time PCR, immunoblotting and immunofluorescence were performed to investigate the molecular mechanisms. Results: Germline loss of the Zbtb38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. Heterozygous loss of Zbtb38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Although this early lethal phenotype, Zbtb38 is dispensable for ES cell establishment and identity. Conclusions: These findings indicate that Zbtb38 is essential for early embryonic development via the suppression of Nanog and Sox2 expression.

Mammalian DNA methylation is an epigenetic modification which is involved in various biological processes, including gene expression regulation. In mice, methyltransferases are responsible for DNA methylation, which are critical for early embryogenesis. However, the significance of methyl-CpG binding proteins (MBPs) that bind methylated CpG remains largely unknown. We previously demonstrated that ZBTB38/CIBZ-a zinc finger type of MBP-is required for ES cell proliferation by positively regulating Nanog expression. However, the physiological function of ZBTB38 remains unclear. In this study, we generated conditional ZBTB38 knockout mice using Cre-loxP technology. Unexpectedly, our results showed that germline loss of the ZBTB38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. We found that heterozygous loss of ZBTB38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Despite this lethal phenotype, ZBTB38 is dispensable for ES cell establishment and identity. Together, these findings indicate that ZBTB38 is essential for early embryonic development, providing new insights into the roles of MBP in implantation.

INTRODUCTION The aim of this study was to investigate the association of the HtrA1 rs11200638 polymorphism with neovascular age-related macular degeneration (nAMD) in Indonesia. METHODS This case-control study included 80 patients with nAMD and 85 controls. Demographic parameters and whole blood were collected from each participant. Genomic DNA was extracted and used to assess the rs11200638 genotype by PCR and restriction enzyme digestion. Associations between the HtrA1 rs11200638 polymorphism and other risk factors for susceptibility to nAMD were assessed using the logistic regression model. RESULTS Significant allelic associations between the HtrA1 polymorphism and nAMD were detected (odds ratio [OR] 8.67; 95% confidence interval [CI] 4.88-15.41; P

Tissue fibrosis and organ dysfunction are hallmarks of age-related diseases including heart failure, but it remains elusive whether there is a common pathway to induce both events. Through single-cell RNA-seq, spatial transcriptomics, and genetic perturbation, we elucidate that high-temperature requirement A serine peptidase 3 (Htra3) is a critical regulator of cardiac fibrosis and heart failure by maintaining the identity of quiescent cardiac fibroblasts through degrading transforming growth factor-β (TGF-β). Pressure overload downregulates expression of Htra3 in cardiac fibroblasts and activated TGF-β signaling, which induces not only cardiac fibrosis but also heart failure through DNA damage accumulation and secretory phenotype induction in failing cardiomyocytes. Overexpression of Htra3 in the heart inhibits TGF-β signaling and ameliorates cardiac dysfunction after pressure overload. Htra3-regulated induction of spatio-temporal cardiac fibrosis and cardiomyocyte secretory phenotype are observed specifically in infarct regions after myocardial infarction. Integrative analyses of single-cardiomyocyte transcriptome and plasma proteome in human reveal that IGFBP7, which is a cytokine downstream of TGF-β and secreted from failing cardiomyocytes, is the most predictable marker of advanced heart failure. These findings highlight the roles of cardiac fibroblasts in regulating cardiomyocyte homeostasis and cardiac fibrosis through the Htra3-TGF-β-IGFBP7 pathway, which would be a therapeutic target for heart failure.

Supanji Supanji,1– 4 Dewi Fathin Romdhoniyyah,1,2 Muhammad Bayu Sasongko,1,2,4 Angela Nurini Agni,1,2,4 Firman Setya Wardhana,1,2,4 Tri Wahyu Widayanti,1,2,4 Muhammad Eko Prayogo,1,2,4 Ayudha Bahana Ilham Perdamaian,1,2 Aninditta Dianratri,1,2 Masashi Kawaichi,5 Chio Oka5 1Department of Ophthalmology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia; 2Department of Ophthalmology, Dr. Sardjito General Hospital, Yogyakarta, Indonesia; 3Ophthalmology Clinic, Military Air Force Central Hospital Dr. Suhardi Hardjolukito, Yogyakarta, Indonesia; 4Ophthalmology Clinic, Dr YAP Eye Hospital, Yogyakarta, Indonesia; 5Laboratory of Gene Function in Animals, Nara Institute of Science and Technology, Ikoma, Nara, JapanCorrespondence: Supanji SupanjiDepartment of Ophthalmology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Jalan Farmako Sekip Utara, Yogyakarta, 55281, IndonesiaTel +62 274 560300Fax +62 274 581 876Email supanji@ugm.ac.idPurpose: This study aimed to determine the association of ARMS2 A69S, ARMS2 del443ins54, and CFH Y402H polymorphisms with neovascular age-related macular degeneration (nAMD) for the first time in an Indonesian population.Patients and Methods: Our case–control study involved 104 nAMD and 100 control subjects. AMD diagnosis was evaluated by retinal specialists based on color fundus photography and optical coherence tomography. The polymorphisms on CFH Y402H and ARMS2 A69S were analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP), whereas ARMS2 del443ins54 was evaluated by PCR-based assay.Results: Significant allelic associations with nAMD were detected on all polymorphisms (P< 0.05), with stronger association with the ARMS2 A69S (OR 3.13; 95% CI 2.08– 4.71; P< 0.001) and ARMS2 del443ins54 (OR 3.28; 95% CI 2.17– 4.95; P< 0.001) polymorphisms than with CFH Y402H (OR 2.08; 95% CI 1.08– 3.99; P=0.028). Genotype analysis showed a statistical difference between nAMD and the control group for all polymorphisms (P< 0.05). However, the association with nAMD was weaker for CFH Y402H (P=0.043) than for ARMS2 A69S and ARMS2 del443ins54 (P< 0.001). A significant interaction between ARMS2 A69S and hypertension was documented (OR 9.53; 95% CI 3.61– 25.1; P< 0.001).Conclusion: Our findings indicate that ARMS2 A69S and ARMS2 del443ins54 polymorphisms are strongly associated with the risk of nAMD for the first time in an Indonesian population. The risk of nAMD increased when the presence of risk alleles from ARMS2 A69S was combined with the presence of hypertension.Keywords: age-related macular degeneration, ARMS2, CFH, polymorphism

Neovascular Age-related Macular Degeneration (nAMD) is one of the major factors for blindness and impaired visual acuity in elderly people. The aim of this study was CFH gene screening in Age-Related Macular Degeneration patients in Indonesia. This study was performed in 106 AMD patients and 104 controls for genomic markers in the Complement Factor H (CFH). The diagnosis of AMD was carried out by retinal specialists based on color fundus photography and optical coherence tomography. Informed consent was given to patients then proceed to blood sampling and recording of body parameters (BMI, smoking, other systemic diseases). CFH polymorphisms were then analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP). There was no association between genetics polymorphism with nAMD. From the research can be inferred that association between genetics polymorphism with nAMD was insignificant.

Purpose This study aimed to determine the association of ARMS2 A69S, ARMS2 del443ins54, and CFH Y402H polymorphisms with neovascular age-related macular degeneration (nAMD) for the first time in an Indonesian population. Patients and Methods Our case–control study involved 104 nAMD and 100 control subjects. AMD diagnosis was evaluated by retinal specialists based on color fundus photography and optical coherence tomography. The polymorphisms on CFH Y402H and ARMS2 A69S were analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP), whereas ARMS2 del443ins54 was evaluated by PCR-based assay. Results Significant allelic associations with nAMD were detected on all polymorphisms (P

This research aims to investigate the HtrA1 serine protease circulating level of Age-related Macular Degeneration (AMD) patients in Yogyakarta, Indonesia. This study was conducted from January to August 2019 which included 38 AMD patients and 16 Non-AMD patients/controls (two groups). Baseline data and blood sample were collected. ELISA assay was used to measure the HtrA1 serine protease circulating level on both groups. SNP genotyping of rs10490924 was using restriction enzyme digestion. This study used The IBM SPSS® version 24 (Chicago, The USA) to determine the relationship between HtrA1 expression level and AMD incidence. AMD patients had higher HtrA1 serine protease level (35.31) than controls (30.08). However, there is no association found between HtrA1 serine protease level and AMD incidence (p-value>0.05, CI 95 %). However, HtrA1 serine protease did not associate positively to AMD incidence in Yogyakarta samples. Further analysis by grouping AMD patient based on the rs10490924 genotype show no statistical correlation between HTRA1 to the incidence of AMD. This result might be due to the lack of samples in the study groups. Future studies with larger number of samples are advised to better see the association between Htra1 serine protease level and AMD incidence.

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

Vascular smooth muscle cell (VSMC) dysfunction is a hallmark of small vessel disease, a common cause of stroke and dementia. Two of the most frequently mutated genes in familial small vessel disease are HTRA1 and NOTCH3. The protease HTRA1 cleaves the NOTCH3 ligand JAG1 implying a mechanistic link between HTRA1 and Notch signaling. Here we report that HTRA1 is essential for VSMC differentiation into the contractile phenotype. Mechanistically, loss of HTRA1 increased JAG1 protein levels and NOTCH3 signaling activity in VSMC. In addition, the loss of HTRA1 enhanced TGFβ-SMAD2/3 signaling activity. Activation of either NOTCH3 or TGFβ signaling resulted in increased transcription of the HES and HEY transcriptional repressors and promoted the contractile VSMC phenotype. However, their combined over-activation led to an additive accumulation of HES and HEY proteins, which repressed the expression of contractile VSMC marker genes. As a result, VSMC adopted an immature phenotype with impaired arterial vasoconstriction in Htra1-deficient mice. These data demonstrate an essential role of HTRA1 in vascular maturation and homeostasis by controlling Notch and TGFβ signaling.

Preserving the mature articular cartilage of joints is a critical focus in the prevention and treatment of osteoarthritis. We determined whether the genetic inactivation of high-temperature requirement A1 (HtrA1) can significantly attenuate the degradation of articular or condylar cartilage. Two types of mouse models of osteoarthritis were used, a spontaneous mutant mouse model [type XI collagen-haploinsufficient (Col11a1+/-) mice] and two post-traumatic mouse models [destabilization of the medial meniscus (DMM) on the knee and a partial discectomy (PDE) on the temporomandibular joint]. Three different groups of mice were generated: i) HtrA1 was genetically deleted from Col11a1+/- mice (HtrA1-/-;Col11a1+/-), ii) HtrA1-deficient mice (HtrA1-/-) were subjected to DMM, and iii) HtrA1-/- mice were subjected to PDE. Knee and temporomandibular joints from the mice were characterized for evidence of cartilage degeneration. The degradation of articular or condylar cartilage was significantly delayed in HtrA1-/-;Col11a1+/- mice and HtrA1-/- mice after DMM or PDE. The amount of collagen type VI was significantly higher in the articular cartilage in HtrA1-/-;Col11a1+/- mice, compared with that in Col11a1+/- mice. The genetic removal of HtrA1 may delay the degradation of articular or condylar cartilage in mice.

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 in vitro 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.

Abnormal levels of High temperature requirement A1 (HtrA1) protein have been repeatedly observed in sera and placentas of preeclampsia patients. To understand the functions of HtrA1 in placentation and in the etiology of preeclampsia, we established HtrA1−/− mice. HtrA1−/− mice show intrauterine growth retardation, and their placentas are small due to a reduced size of the junctional zone and aberrant vascularization in the labyrinth at the mid-gestation stage. HtrA1 is expressed by Tpbpa-positive trophoblast precursors in the outer ectoplacental cone and junctional zone from embryonic day 7.5 to 10.5. In the HtrA1−/− placenta, Tpbpa-positive cell precursors are decreased in the early stage. Spongiotrophoblasts and glycogen trophoblast cells, both of which differentiate from Tpbpa-positive precursors, are consequently decreased in the junctional zone. Fewer spiral artery-associated trophoblast giant cells, another cell type derived from Tpbpa-positive precursors, invade the decidua and associate with maternal arteries in the HtrA1−/− placenta than in the wild type placenta. Maternal arteries in the HtrA1−/− decidua have narrower lumens, thicker arterial walls, and more vascular smooth muscle cells remaining in the walls than those in the wild type decidua, indicating impaired remodeling of maternal arteries. These results indicate that HtrA1 plays important roles in the differentiation of trophoblasts from Tpbpa-positive precursors in the ectoplacental cone. Insufficient levels of HtrA1 cause poor placental development and intrauterine growth retardation, due to aberrant trophoblast differentiation and consequent defects in maternal artery remodeling, and may contribute to the onset of preeclampsia.

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

To examine the versatility of the trans-bisglycoconjugation architecture for 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TFPP) derivative-based photodynamic therapy (PDT), 25 porphyrin derivat...

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

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly. Wet AMD includes typical choroidal neovascularization (CNV) and polypoidal choroidal vasculopathy (PCV). The etiology and pathogenesis of CNV and PCV are not well understood. Genome-wide association studies have linked a multifunctional serine protease, HTRA1, to AMD. However, the precise role of HTRA1 in AMD remains elusive. By transgenically expressing human HTRA1 in mouse retinal pigment epithelium, we showed that increased HTRA1 induced cardinal features of PCV, including branching networks of choroidal vessels, polypoidal lesions, severe degeneration of the elastic laminae, and tunica media of choroidal vessels. In addition, HTRA1 mice displayed retinal pigment epithelium atrophy and photoreceptor degeneration. Senescent HTRA1 mice developed occult CNV, which likely resulted from the degradation of the elastic lamina of Bruch's membrane and up-regulation of VEGF. Our results indicate that increased HTRA1 is sufficient to cause PCV and is a significant risk factor for CNV.

Deficiency of caytaxin results in hereditary ataxia or dystonia in humans, mice and rats. Our yeast two-hybrid screen identified kinesin light chains (KLCs) as caytaxin-binding proteins. The tetratricopeptide-repeat region of KLC1 recognizes the ELEWED sequence (amino acids 115-120) of caytaxin. This motif is conserved among BNIP-2 family members and other KLC-interacting kinesin cargo proteins such as calsyntenins. Caytaxin associates with kinesin heavy chains (KHCs) indirectly by binding to KLCs, suggesting that caytaxin binds to the tetrameric kinesin molecule. In cultured hippocampal neurons, we found that caytaxin is distributed in both axons and dendrites in punctate patterns, and it colocalizes with microtubules and KHC. GFP-caytaxin expressed in hippocampal neurons is transported at a speed (∼1 μm/second) compatible with kinesin movement. Inhibition of kinesin-1 by dominant-negative KHC decreases the accumulation of caytaxin in the growth cone. Caytaxin puncta do not coincide with vesicles containing known kinesin cargos such as APP or JIP-1. A part of caytaxin, however, colocalizes with mitochondria and suppression of caytaxin expression by RNAi redistributes mitochondria away from the distal ends of neurites. These data indicate that caytaxin binds to kinesin-1 and functions as an adaptor that mediates intracellular transport of specific cargos, one of which is the mitochondrion.

trans-Bisthioglycosylated tetrakis(fluorophenyl)chlorin (7) was designed as a powerful photodynamic therapy (PDT) photosensitizer based on the findings of our systematic studies. We show here that the trans-bisthioglycosylated structure of 7 enhanced its uptake by HeLa cells and that the chlorin ring of 7 increased the efficiency of reactive oxygen species generation under the standard condition of our photocytotoxicity test. The versatility of 7 in PDT treatment was established using weakly metastatic B16F1 melanoma cells, metastatic 4T1 breast cancer cells, the RGK-1 gastric carcinoma mucosal cell line, and three human glioblastoma cell lines (U87, U251, and T98G). The pharmacokinetics of 7 in mice bearing 4T1 breast cancer cells showed a high tumor-to-skin concentration ratio (approximately 60) at 24 h after intraperitoneal injection. The PDT efficacy of 7 in vivo was approximately 250-times higher than that of mono-l-aspartyl chlorin e6 (9) in mice bearing 4T1 breast cancer cells.

HtrA1, a member of the mammalian HtrA (high temperature requirement A) serine protease family, has a highly conserved protease domain followed by a PDZ domain. Accumulating evidence has indicated that PDZ domains regulate protease activity of HtrA proteins. We searched for binding partners of the PDZ domain of mouse HtrA1 by yeast two-hybrid screening, and isolated proteins that were recognized by the HtrA1 PDZ domain through their C-terminal ends with a core consensus Phi-X-Phi-[V/L/F/A]-COOH sequence (where Phi is a hydrophobic/non-polar amino acid). C-propeptides of fibrillar collagens were most frequently isolated. Type III procollagen alpha1 C-propeptide, which was used as a model protein, was digested by HtrA1. HtrA1 cleavage of the collagen C-propeptide was enhanced by reductive denaturation of the C-propeptide and partly inhibited by removal of the C-terminal four amino acids from the C-propeptide, suggesting that the substrate recognition was facilitated by the binding of the free C-terminal ends of substrates to the PDZ domain of HtrA1. The synthetic oligopeptide (GM130Pep) that fitted the consensus recognition sequence bound to HtrA1 with a high affinity (K(d)=6.0 nM). GM130Pep stimulated HtrA1 protease activity 3- to 4-fold, but did not efficiently stimulate the activity of an HtrA1 mutant lacking the PDZ domain, supporting the notion that the PDZ domain enhances protease activity upon ligand binding. The peptide derived from Type III collagen alpha1 C-propeptide specifically stimulated protease activity of HtrA1, but did not stimulate nor significantly bind to HtrA2, suggesting that the collagen C-propeptide is a specific physiological regulator of HtrA1.

HtrA1, a member of the mammalian HtrA serine protease family, has a highly conserved protease domain followed by a PDZ domain. Because HtrA1 is a secretory protein and has another functional domain with homology to follistatin, we examined whether HtrA1 functions as an antagonist of Tgfbeta family proteins. During embryo development, mouse HtrA1 was expressed in specific areas where signaling by Tgfbeta family proteins plays important regulatory roles. The GST-pulldown assay showed that HtrA1 binds to a broad range of Tgfbeta family proteins, including Bmp4, Gdf5, Tgfbetas and activin. HtrA1 inhibited signaling by Bmp4, Bmp2, and Tgfbeta1 in C2C12 cells, presumably by preventing receptor activation. Experiments using a series of deletion mutants indicated that the binding activity of HtrA1 required the protease domain and a small linker region preceding it, and that inhibition of Tgfbeta signaling is dependent on the proteolytic activity of HtrA1. Misexpression of HtrA1 near the developing chick eye led to suppression of eye development that was indistinguishable from the effects of noggin. Taken together, these data indicate that HtrA1 protease is a novel inhibitor of Tgfbeta family members.

The transcription factor recombination signal sequence-binding protein Jκ (RBP-J) is a key downstream element in the signaling pathway of all four mammalian Notch receptors that are critically involved in the control of embryonic and adult development. RBP-J-deficient mice display complex defects and die around day 9.5 postcoitum. Here, we investigate the function of RBP-J in the development of mesodermal cell lineages by using the OP9 stroma coculture system. RBP-J-deficient embryonic stem (ES) cells gave rise to cardiomyocytes, endothelial cells, and primitive and definitive hematopoietic cells. Thus, RBP-J-mediated signals are not required for generation of these cell types. However, when compared with parental RBP-J-expressing ES cells, cardiomyogenesis derived from RBP-J-deficient ES cells was increased. Repression over the cardiogenic pathway was restored by expressing RBP-J in RBP-J-deficient ES cells. Our data indicate that Notch signaling via RBP-J plays an important role for the correct specification of myocardial cell fates.

Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, whereas Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 (age-related maculopathy susceptibility 2) and HTRA1 (high temperature requirement A serine peptidase 1) genes. Transcription regulator activity was localized in mouse retinas using heterozygous HtrA1 knock-out mice in which HtrA1 exon 1 was replaced with β-galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein that bound the insertion/deletion sequence, LYRIC (lysine-rich CEACAM1 co-isolated) protein. In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant up-regulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained up-regulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HtrA1 and exhibits characteristics similar to those of wet AMD patients. Background: The biological function of insertion/deletion sequences associated with AMD has not been fully characterized. Results: The HTRA1 regulatory region contains an insertion/deletion sequence that is significantly up-regulated in retinal neuronal cell lines. Conclusion: HTRA1 expression is enhanced by a mutation in the insertion/deletion in the HTRA1 regulatory region. Significance: This is the characterization of the HTRA1 regulatory elements and the effect of insertion/deletion sequences associated with AMD.

BNIP-2 shows high homology with the Cayman ataxia protein, caytaxin, which functions as a kinesin-1 adapter bridging cargos and kinesin light chains (KLCs). BNIP-2 is known to induce cell shape changes when over-expressed in culture cells, but its physiological functions are mostly unknown. BNIP-2 interacts with KLC through the conserved WED motif in the N-terminal region of BNIP-2. Interaction with KLC and transportation by kinesin-1 are essential for over-expressed BNIP-2 to elongate cells and induce cellular processes. Endogenous BNIP-2 localizes to the Golgi apparatus, early and recycling endosomes and mitochondria, aligned with microtubules, and moves at a speed compatible with kinesin-1 transportation. The CRAL-TRIO domain of BNIP-2 specifically interacts with phosphatidylserine, and the vesicular localization of BNIP-2 requires interaction with this phospholipid. BNIP-2 mutants which do not bind phosphatidylserine do not induce morphological changes in cells. These data show that similar to caytaxin, BNIP-2 is a kinesin-1 adapter involved in vesicular transportation in the cytoplasm and that association with cargos depends on interaction of the CRAL-TRIO domain with membrane phosphatidylserine.

Background Development of the pancreas and the nervous tissues is regulated by common transcription factors. A basic helix-loop-helix protein, p48 of pancreas transcription factor 1 (PTF1), is essential for differentiation of the exocrine acinar cells. Results We isolated PTF1 p48 from 9.5-day mouse embryos as a binding protein of RBP-Jκ, a mediator of Notch signalling. p48 bound to RBP-Jκ more strongly than and in a distinct way from Notch1. In 9.5–12.5 day embryos, p48 was expressed in the dorsal part of the neural tube as well as in the pancreatic buds. Two lines of evidence suggested functions of p48 in neurogenesis: (i) expression of p48 was induced in P19 cells when they committed to neural fate upon retinoic acid treatment, and (ii) p48 over-expressed in Xenopus embryos repressed the development of neuronal precursors. p48 inhibited the MASH1-activated transcription from the E-box, while p48 stimulated transcription from the PTF1 motif synergistically with E47. The p48/E47-activated transcription from the PTF1 motif was stimulated further by RBP-Jκ and RBP-Jκ derivatives that mimicked the active RBP-Jκ/Notch complex. Conclusions In developing embryos, p48 is expressed in both the nervous system and the pancreas. p48 inhibits neuronal differentiation. We propose possible mechanisms for this inhibition.

RBP-Jkappa is a novel type of transcriptional regulatory protein that does not contain any known DNA-binding motif. We raised anti-RBP-Jkappa monoclonal antibodies (K0043 and T6709) to investigate the roles of RBP-Jkappa in cell differentiation. These antibodies stained nuclei of undifferentiated embryonic stem cells and F9 cells but not those of the other differentiated cell lines tested so far although the RBP-Jkappa protein is expressed at similar levels. Interestingly, differentiated F9 cells lost the immunostaining reaction with the anti-RBP-Jkappa monoclonal antibodies. Biochemical subcellular fractionation study showed that the majority of RBP-Jkappa was localized in nuclei of F9 cells and that there are at least two forms of the RBP-Jkapppa protein in the nuclei of undifferentiated F9 cells, a free form and a chromatin-bound form. Upon induction of F9 cell differentiation, free nuclear RBP-Jkappa disappeared concomitantly with the loss of immunostaining, suggesting that the anti-RBP-Jkapppa antibodies cannot recognize chromatin-bound RBP-Jkappa. Since there is no evidence to indicate covalent modification of RBP-Jkappa, we assume that chromatin-bound RBP-Jkappa interacts with a large number of proteins which block the exposure of RBP-Jkappa epitopes to the monoclonal antibodies.

We have constructed transgenic mice carrying an artificial substrate of V(D)J recombination. In this substrate, the only DNA fragments derived from Ig genes were short stretches of recombination signal sequences. This artificial substrate was rearranged at high frequency in lymphocytes, although in non-lymphoid cells no rearrangement was detected even by a sensitive PCR assay. This result indicates that the V(D)J recombination requires only the signal sequences and that a recombination similar to the V(D)J recombination does not occur in non-lymphoid tissues including the central nervous tissue. A protein binding to the V(D)J recombination signals was purified and its cDNA was cloned. This protein, termed RBP-J kappa, was initially considered to be involved in V(D)J recombination because of its DNA binding specificity and structural similarity to site-specific recombinases known as the integrase family. However, further study on the Drosophila homolog of RBP-J kappa indicated that RBP-J kappa probably functions as a transcription factor in the differentiation of the peripheral nervous tissues. The exact function of RBP-J kappa is still unknown. Analogous to the Drosophila gene, it is suggested that mouse RBP-J kappa participates in the regulation of differentiation of various tissues.

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 p21(CIP1/WAF1) and p16(INK4a), 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.

Palladium(II) complexes of glycoconjugated porphyrin and pyrrolidine-fused chlorin were prepared to examine sugar and heavy atom effects on in vitro photocytotoxicity. Cellular uptake into HeLa cells was enhanced by introducing sugar units regardless of other features, such as the central ion (free base or palladium(II) ion) and the ring structure (porphyrin or chlorin). The palladium(II) complex of glycoconjugated pyrrolidine-fused chlorin (PdPC2) exerted an excellent degree of photocytotoxicity not only on HeLa cells, but also on metastatic B16-BL6 cells, weakly metastatic B16F1 cells, and metastatic 4T1 cells. However, free-base glycoconjugated pyrrolidine-fused chlorin (PC2) also exerted similar or much higher photocytotoxicity rather than PdPC2. Therefore, the palladium(II) ion did not improve the in vitro photocytotoxicity of PC2. The enhanced singlet oxygen generation of palladium(II) complexes (i.e., the heavy atom effect) was confirmed at least in O(2)-saturated D(2)O. In addition, the formation of hydrogen peroxide and hydroxyl radical were also detected in O(2)-saturated phosphate buffered saline. However, the reactive oxygen species (ROS) generation efficiency, which is the product of the (relative) quantum yield of each ROS and the light absorbing ability, did not fit the trends of photocytotoxicity seen for the photosensitizers. In our glycoconjugated photosensitizers tested, the best indicator of the photocytotoxicity was found to be the light absorbing ability (namely, the oscillator strength in the wavelength region applied in the photocytotoxicity test). These results indicated that photochemical characteristics of glycoconjugated photosensitizers were notably susceptible to the microenvironment. The biological characteristics, such as the sugar effect, were a much more reliable approach to improving the photocytotoxicity of photosensitizers.

We have isolated a cDNA clone (RBP-2) for the protein (RBP-J kappa) which binds to immunoglobulin recombination signals with 23-base pair spacers (Matsunami, N., Hamaguchi, Y., Yamamoto, Y., Kuze, K., Kangawa, K., Matsuo, H., Kawaichi, M., and Honjo, T. (1989) Nature 342, 934-937). During further screening of a cDNA library from the same mouse pre-B cell line (38B9), we have isolated a second cDNA clone (RBP-2N) which differs from RBP-2 in its 5' sequence. RNase protection assays indicated that the RBP-2N type mRNA was produced in 10-20 times the quantity as RBP-2 mRNA. To elucidate the relationship between these two mRNAs, we analyzed the genomic organization of the RBP-J kappa gene. Southern hybridization of mouse genomic DNA detected at least 7 EcoRI fragments hybridizing to an RBP-2 cDNA probe, suggesting a complex structure for the RBP-J kappa gene. Cloning of each EcoRI fragment revealed one functional RBP-J kappa gene and three related genes. The functional gene was composed of 11 exons and spanned at least 50 kilobase pairs. The sequence of exon 1 and its 5'-flanking region contained a GC-rich promoter-like region but no apparent TATA box. The initiation site of transcription was heterogeneous, and the two types of mRNA are produced from the same exon by transcription initiation at different sites and by different usage of splice signals. Two of the three related genes were processed pseudogenes with scattered stop codons. The other was also a processed gene with a sequence exactly the same as that of RBP-2, except that this gene lacked the sequence corresponding to the first exon of the functional gene.

We have constructed a plasmid, pLTR100, which contains human interleukin 2 receptor light (IL-2R L) chain cDNA in the inverted orientation relative to the upstream SV40 promoter. The cDNA segment is flanked by the immunoglobulin gene recombination signal sequences so that the cDNA segment can invert and the human IL-2R L chain is subsequently expressed under the control of the SV40 promoter. A murine pre-B cell line, 38B9, transfected with pLTR100 began to express the human IL-2R L chain on the cell surface. The frequency of human IL-2R L chain positive cells increased almost linearly up to 50% for 60 days of culture after transfection. Southern blot analysis and sequencing of the DNA fragments at the recombination junction confirmed that the cDNA segment was inverted in a signal sequence-dependent manner by the variable-diversity-joining recombination process. Transgenic mice bearing the recombination substrate DNA similar to pLTR100 expressed the human IL-2 L chain in the spleen, thymus, and bone marrow, but not in the other tissues examined at the detectable level. Both IgM- and CD3-positive cells expressed the human IL-2R L chain, indicating that this artificial DNA can serve as a substrate for recombination both in B- and T-cells and that another DNA segment may be necessary to confer the cell-type specificity on the substrate DNA.

Recent studies show LDL receptor-related protein 1B, LRP1B as a transducer of extracellular signals. Here, we identify six interacting partners of the LRP1B cytoplasmic region by yeast two-hybrid screen and confirmed their in vivo binding by immunoprecipitation. One of the partners, PICK1 recognizes the C-terminus of LRP1B and LRP1. The cytoplasmic domains of LRP1B are phosphorylated by PKCα about 100 times more efficiently than LRP1. Binding of PICK1 inhibits phosphorylation of LRP1B, but does not affect LRP1 phosphorylation. This study presents the possibility that LRP1B participates in signal transduction which PICK1 may regulate by inhibiting PKCα phosphorylation of LRP1B. Structured summary MINT-6801075: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with SNTG2 (uniprotkb:Q925E0) by two hybrid (MI:0018) MINT-6801030, MINT-6801468: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by two hybrid (MI:0018) MINT-6801284: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by anti tag coimmunoprecipitation (MI:0007) MINT-6801108: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Grb7 (uniprotkb:Q03160) by two hybrid (MI:0018) MINT-6801090: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by two hybrid (MI:0018) MINT-6801008: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by two hybrid (MI:0018) MINT-6801052: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-2 (uniprotkb:Q9ERE9) by two hybrid (MI:0018) MINT-6801258, MINT-6801271: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by anti tag coimmunoprecipitation (MI:0007) MINT-6801244: RanBPM (uniprotkb:P69566) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007) MINT-6801131, MINT-6801158: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by anti tag coimmunoprecipitation (MI:0007) MINT-6801231: PICK1 (uniprotkb:Q80VC8) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007) MINT-6801173: Jip-1b (uniprotkb:Q9WVI9-1) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)

HtrA which is characterized by the combination of a trypsin-like catalytic domain with at least one C-terminalPDZ domain is a highly conserved family of serine proteases found in a wide range of organisms. However theidentified HtrA family numbers varies among spesies, for example the number of mammalian, Eschericia coli,fruit fly-HtrA family are 4, 3 and 1 gene respectively. One gene is predicted exist in zebrafish. Since no completeinformation available on zebrafish HtrA, in this paper zebrafish HtrA (zHtrA) gene was analyzed. The zHtrA isbelonged to HtrA1 member and predicted encodes 478 amino acids with a signal peptide, a IGF binding domain,a Kazal-type inhibitor domain in the up stream of HtrA-bacterial homolog. At the amino acid sequence the zHtrA1showed the 69%, 69%, 68%, 54% and 54% with the rat HtrA1, mouse HtrA1, human HtrA1, human HtrA3 andmouse HtrA4 respectively. The zHtrA1 is firstly expressed at 60 hpf and mainly in the vertebral rudiments in thetail region.

Levels of HtrA1 protein in cartilage have been reported to elevate in joints of human osteoarthritis patients. To understand roles of HtrA1 in normal osteogenesis as well as in pathogenesis of arthritis, we examine HtrA1 expression pattern during bone and cartilage development and in articular cartilage affected by experimental arthritis. HtrA1 is not expressed in mesenchymal or cartilage condensations before initiation of ossification. When ossification begins in the condensations, the expression of HtrA1 starts in chondrocytes undergoing hypertrophic differentiation near the ossification center. Hypertrophic chondrocytes found in adult articular cartilage and epiphyseal growth plates also express HtrA1. When arthritis is induced by injection of anti-collagen antibodies and lipopolysaccharide, resting chondrocytes proceed to terminal hypertrophic differentiation and start expressing HtrA1. These data suggest that hypertrophic change induces HtrA1 expression in chondrocytes both in normal and pathological conditions. HtrA1 has been reported to inhibit TGF-beta signaling. We show that HtrA1 digests major components of cartilage, such as aggrecan, decorin, fibromodulin, and soluble type II collagen. HtrA1 may, therefore, promote degeneration of cartilage by inducing terminal hypertrophic chondrocyte differentiation and by digesting cartilage matrix though its TGF-beta inhibitory activity and protease activity, respectively. In bone, active cuboidal osteoblasts barely express HtrA1, but osteoblasts which flatten and adhere to the bone matrix and osteocytes embedded in bone are strongly positive for HtrA1 production. The bone matrix shows a high level of HtrA1 protein deposition akin to that of TGF-beta, suggesting a close functional interaction between TGF-beta and HtrA1.

The expression of mouse HtrA1 is developmentally regulated and restricted in embryo tissues which depend largely on TGF-beta signaling for their differentiation. We examined whether mouse HtrA3, another HtrA family member very close to HtrA1, shows similar expression patterns. HtrA3 and -1 were expressed mostly in the same embryonic organs but exhibited complementary patterns in various tissues; the lens epithelial cells in day 12.5 embryo expressed HtrA3 whereas the ciliary body and pigment retina expressed HtrA1. In the vertebrae of day 14.5 embryo, HtrA3 was expressed in the tail region, but HtrA1 was predominantly expressed in the thoracic and lumbar regions. Similar to HtrA1, HtrA3 bound to various TGF-beta proteins and inhibited the signaling of BMP-4, -2 and TGF-beta 1. HtrA3 did not inhibit signaling originated from a constitutively active BMP receptor, indicating that the inhibition occurred upstream of the cell surface receptor. HtrA3 also showed proteolytic activities indistinguishable from those of HtrA1 toward beta-casein and some extracellular matrix (ECM) proteoglycans. The protease activity was absolutely required for the TGF-beta signal inhibition activity. All these data suggest that HtrA3 and -1 have the overlapping biological activities but can function in complementary fashion in certain types of tissues.