Candesartan prevents arteriopathy progression in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy model.

Cerebral small vessel disease (CSVD) causes dementia and gait disturbance due to arteriopathy. Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a hereditary form of CSVD caused by loss of high-temperature requirement A1 (HTRA1) serine protease activity. In CARASIL, arteriopathy causes intimal thickening, smooth muscle cell (SMC) degeneration, elastic lamina splitting, and vasodilation. The molecular mechanisms were proposed to involve the accumulation of matrisome proteins as substrates or abnormalities in transforming growth factor ß (TGF-ß) signaling. Here, we show that HTRA1-/- mice exhibited features of CARASIL-associated arteriopathy: intimal thickening, abnormal elastic lamina, and vasodilation. In addition, the mice exhibited reduced distensibility of the cerebral arteries and blood flow in the cerebral cortex. In the thickened intima, matrisome proteins, including the hub protein fibronectin (FN) and latent TGF-ß binding protein 4 (LTBP-4), which are substrates of HTRA1, accumulated. Candesartan treatment alleviated matrisome protein accumulation and normalized the vascular distensibility and cerebral blood flow. Furthermore, candesartan reduced the mRNA expression of Fn1, Ltbp-4, and Adamtsl2, which are involved in forming the extracellular matrix network. Our results indicate that these accumulated matrisome proteins may be potential therapeutic targets for arteriopathy in CARASIL.

Cell density-dependent proteolysis by HtrA1 induces translocation of zyxin to the nucleus and increased cell survival.

Proteases modulate critical processes in cutaneous tissue repair to orchestrate inflammation, cell proliferation and tissue remodeling. However, the functional consequences and implications in healing impairments of most cleavage events are not understood. Using iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) we had characterized proteolytic signatures in a porcine wound healing model and identified two neo-N termini derived from proteolytic cleavage of the focal adhesion protein and mechanotransducer zyxin. Here, we assign these proteolytic events to the activity of either caspase-1 or serine protease HtrA1 and analyze the biological relevance of the resultant zyxin truncations. By cellular expression of full-length and truncated zyxin proteins, we demonstrate nuclear translocation of a C-terminal zyxin fragment that could also be generated in vitro by HtrA1 cleavage and provide evidence for its anti-apoptotic activities, potentially by regulating the expression of modulators of cell proliferation, protein synthesis and genome stability. Targeted degradomics correlated endogenous generation of the same zyxin fragment with increased cell density in human primary dermal fibroblasts. Hence, this newly identified HtrA1-zyxin protease signaling axis might present a novel mechanism to transiently enhance cell survival in environments of increased cell density like in wound granulation tissue.

Loss of the serine protease HTRA1 impairs smooth muscle cells maturation.

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 TGFß-SMAD2/3 signaling activity. Activation of either NOTCH3 or TGFß 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 TGFß signaling.

HTRA1-Dependent Cell Cycle Proteomics.

The HTRA1 gene encoding an evolutionary conserved protein quality-control factor can be epigenetically silenced or inactivated by mutation under pathologic conditions such as cancer. Recent evidence suggests that the loss of HTRA1 function causes multiple phenotypes, including the acceleration of cell growth, delayed onset of senescence, centrosome amplification, and polyploidy, suggesting an implication in the regulation of the cell cycle. To address this model, we performed a large-scale proteomics study to correlate the abundance of proteins and HTRA1 levels in various cell cycle phases using label-free-quantification mass spectrometry. These data indicate that the levels of 4723 proteins fluctuated in a cell-cycle-dependent manner, 2872 in a HTRA1-dependent manner, and 1530 in a cell-cycle- and HTRA1-dependent manner. The large number of proteins affected by the modulation of HTRA1 levels supports its general role in protein homeostasis. Moreover, the detected changes in protein abundance, in combination with pull-down data, implicate HTRA1 in various cell cycle events such as DNA replication, chromosome segregation, and cell-cycle-dependent apoptosis. These results highlight the wide implications of HTRA1 in cellular physiology.

Loss of HtrA1 serine protease induces synthetic modulation of aortic vascular smooth muscle cells.

Homozygous mutations of human HTRA1 cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HtrA1-/- mice were examined for arterial abnormalities. Although their cerebral arteries were normal, the thoracic aorta was affected in HtrA1-/- mice. The number of vascular smooth muscle cells (VSMCs) in the aorta was increased in HtrA1-/- mice of 40 weeks or younger, but decreased thereafter. The cross-sectional area of the aorta was increased in HtrA1-/- mice of 40 weeks or older. Aortic VSMCs isolated from HtrA1-/- mice rapidly proliferated and migrated, produced high MMP9 activity, and were prone to oxidative stress-induced cell death. HtrA1-/- VSMCs expressed less smooth muscle α-actin, and more vimentin and osteopontin, and responded to PDGF-BB more strongly than wild type VSMCs, indicating that HtrA1-/- VSMCs were in the synthetic phenotype. The elastic lamina was disrupted, and collagens were decreased in the aortic media. Calponin in the media was decreased, whereas vimentin and osteopontin were increased, suggesting a synthetic shift of VSMCs in vivo. Loss of HtrA1 therefore skews VSMCs toward the synthetic phenotype, induces MMP9 expression, and expedites cell death. We propose that the synthetic modulation is the primary event that leads to the vascular abnormalities caused by HtrA1 deficiency.

High temperature requirement factor A1 (HTRA1) regulates the activation of latent TGF-ß1 in keloid fibroblasts.

Scar treatments are considered a major issue in the plastic surgery field. Activation of the transforming growth factor-ß (TGF-ß)-mediated signaling pathway plays a key role in the scar pathogeneses, and high temperature requirement factor A1 (HTRA1) inhibits TGF-ß1 activation in tumor cells. Our study aims to investigate the role of HTRA1 in the pathogenesis of scars. The mRNA levels of HTRA1 was evaluated by real time PCR, HTRA1 protein expression was determined using western blot and immunohistochemistry, and a luciferase assay was applied to measure dynamic changes of TGF-ß1 activity. We found that the expression of HTRA1 was significantly elevated in keloid tissues, compared to normal skin, and TGF-ß1 mRNA levels slightly increase in the keloid tissue. Furthermore, active TGF-ß1 protein levels and Smad2 phosphorylation significantly increased in the keloid tissue. Treatment with the latent TGF-ß1 or recombinant human HTRA1 (rhHTRA1), alone or in combination, increased Smad2 phosphorylation levels in keloid fibroblasts and active TGF-ß1 contents of associated supernatants. Our results suggest that HTRA1 is involved in the pathogenesis of scars through regulating activation of latent TGF-ß1 in keloid fibroblasts, and our study reveals that HTRA1 is a novel target that regulates scar formation.

High-Temperature Requirement A1 (Htra1) - A Novel Regulator of Canonical Wnt Signaling.

Different cancer types as well as many other diseases are caused by aberrant activation of the canonical Wnt signal transduction pathway, and it is especially implicated in the development and progression of colorectal cancer (CRC). The main effector protein of the canonical Wnt signaling cascade is ß-catenin, which binds to the T- cell factor/lymphoid enhancer factor (TCF/LEF) and triggers the activation of Wnt target genes. Here, we identify the serine protease High-Temperature Requirement A1 (HTRA1) as a novel component of the canonical Wnt pathway. We show that the HTRA1 protein inhibits the Wnt/ß-catenin signaling, in both paracrine and autocrine manners, and affects the expression of several Wnt target genes. Moreover, HTRA1 forms a complex with ß-catenin and reduces the proliferation rates of cells. Taken together, our findings indicate that HTRA1 functions as a novel suppressor of the canonical Wnt signaling pathway.