HTRA1: Ein Kandidatengen für die Altersbedingte Makuladegeneration?

Polymorphismen in der chromosomalen Region 10q26 sind stark mit einem erhöhten Erkrankungsrisiko der Altersabhängigen Makuladegeneration (AMD) assoziiert. In diesem Bereich liegt das Gen high temperature requirement protein A1 (HTRA1). Das Gen kodiert eine Serinprotease, die vor allem in der Qualitätskontrolle von Extrazellulärmatrix (EZM)-Proteinen eine Rolle spielt. Der single nucleotide polymorphism (SNP) rs11200638 im Promotor des HTRA1-Gens verursacht eine Überexpression von HTRA1 im retinalen Pigmentepithel (RPE) und erhöht das Erkrankungsrisiko der AMD deutlich. In AMD-Patienten wurde eine zwei- bis dreifache Überexpression an HTRA1-Protein in RPE-Zellen nachgewiesen. Bislang gab es jedoch keine funktionellen Studien zur Überexpression von HTRA1 im RPE, die Aufschluss über die Beteiligung von HTRA1 an der Entstehung der AMD geben könnten. In dieser Arbeit wurde die Rolle der HTRA1-Überexpression in der Pathogenese der AMD untersucht. Dazu wurden transgene Mäuse generiert, die HTRA1-Protein im RPE 2,7-fach überexprimierten. Die Htra1-transgenen Mäuse entwickelten keine spontane choroidale Neovaskularisation (CNV) in der Retina. Die CNV-Induktion mittels Laserkoagulation ließ in der Fluoreszenz-Angiographie (FAG), der spektralen optischen Kohärenztomographie (SD-OCT) und histologischen Untersuchungen nicht auf eine erhöhte Angiogenese durch die HTRA1-Überexpression schließen. Ebenfalls besaßen Htra1-transgene Mäuse im Vergleich zum Wildtypen keine Expressionsunterschiede vom transformierenden Wachstumsfaktor-beta (TGF-ß), vom Insulin-ähnlichen Wachstumsfaktors-1 (IGF-1) und vom vaskulären endothelialen Wachstumsfaktor (VEGF) im Auge. Unterdessen zeigte sich in Zellkulturüberständen Htra1-transfizierter Nierenzellen (HEK-293-EBNA) eine reduzierte Konzentration von VEGF, Endostatin und Angiogenin im Vergleich zu nicht-transfizierten Zellen und Kontrollen. Die Konzentration von Angiopoietin-1 war dagegen leicht erhöht. Anhand von Transmissonselektronenmikroskopie (TEM)-Bildern war eine Fragmentierung der elastischen Schicht (EL) der Bruchschen Membran (BrM) in Htra1-transgenen Mäusen zu beobachten. Zusätzlich kam es zu verminderten Expressionen von Fibulin-5 und Tropoelastin (TE) in Protein-Lysaten von RPE, BrM und Choroidea. Versuche mit rekombinantem HTRA1 bestätigten weiterhin den Abbau von Fibulin-5 durch HTRA1. Diese Daten deuten auf eine gestörte Elastogenese in der BrM von Htra1-transgenen Mäusen hin. Interessanterweise sind Mutationen im FBLN5 (Fibulin-5)-Gen, die zu einer reduzierten Sekretion von Fibulin-5 führen, mit der AMD assoziiert. Darüber hinaus war die Stabilität der BrM weiterhin durch den HTRA1-bedingten Abbau von Fibronektin gestört. So war die Expression von Fibronektin und Fibronektin-Fragmenten in den transgenen Mäusen erhöht und die Adhäsion Htra1-transfizierter Nieren- und RPE (ARPE-19)-Zellen an Fibronektin deutlich reduziert. Zudem wurden Fibronektin und Nidogen-1 und -2, aber nicht Laminin-1 und Kollagen IV von rekombinantem HTRA1-Protein proteolytisch gespalten. Die Immunfluoreszenzfärbungen auf Paraffinschnitten der Retinae Htra1-transgener und Wildtyp-Mäuse ließen allerdings keine Änderungen in der Farbintensität oder Verteilung von Nidogen-1 und -2 und auch Laminin-1 und Kollagen IV erkennen. Die Ergebnisse dieser Arbeit zeigen, dass die BrM eine wichtige Rolle bei der Entstehung der AMD hat. Eine HTRA1-Überexpression führt durch die Reduktion von Fibulin-5 und TE zu einer gestörten Elastogenese in der EL der BrM. Auf Grund dieser Ergebnisse kann HTRA1 auch mit anderen AMD-Risikogenen wie MMP-9 (Matrixmetalloproteinase-9), TIMP-3 (Gewebeinhibitor von Metalloptroteinasen-3) und FBLN-3, -5 und -6 in Verbindung gebracht werden

Enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) regulate Wnt/beta-catenin-driven trans-differentiation of murine alveolar epithelial cells

The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/beta-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1 alpha (podoplanin;an ATI cell marker),exhibited decreased protein expression upon pharmacological and molecular Wnt/beta-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/beta-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC),whereas ENO1 and PDIA3 along with T1 alpha were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1 alpha expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/beta-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair

BARD1 mediates TGF-β signaling in pulmonary fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-β signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1β, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis. Methods We investigated BARD1 expression as a function of TGF-β in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients. Results FL BARD1 and BARD1β were upregulated in response to TGF-β in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1β in fibrotic tissues. Conclusion Our data suggest that FL BARD1 and BARD1β might be mediators of pleiotropic effects of TGF-β. In particular BARD1β might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment

The ciliopathy gene cc2d2a controls zebrafish photoreceptor outer segment development through a role in Rab8-dependent vesicle trafficking

Ciliopathies are a genetically and phenotypically heterogeneous group of human developmental disorders whose root cause is the absence or dysfunction of primary cilia. Joubert syndrome is characterized by a distinctive hindbrain malformation variably associated with retinal dystrophy and cystic kidney disease. Mutations in CC2D2A are found in ∼10% of patients with Joubert syndrome. Here we describe the retinal phenotype of cc2d2a mutant zebrafish consisting of disorganized rod and cone photoreceptor outer segments resulting in abnormal visual function as measured by electroretinogram. Our analysis reveals trafficking defects in mutant photoreceptors affecting transmembrane outer segment proteins (opsins) and striking accumulation of vesicles, suggesting a role for Cc2d2a in vesicle trafficking and fusion. This is further supported by mislocalization of Rab8, a key regulator of opsin carrier vesicle trafficking, in cc2d2a mutant photoreceptors and by enhancement of the cc2d2a retinal and kidney phenotypes with partial knockdown of rab8. We demonstrate that Cc2d2a localizes to the connecting cilium in photoreceptors and to the transition zone in other ciliated cell types and that cilia are present in these cells in cc2d2a mutants, arguing against a primary function for Cc2d2a in ciliogenesis. Our data support a model where Cc2d2a, localized at the photoreceptor connecting cilium/transition zone, facilitates protein transport through a role in Rab8-dependent vesicle trafficking and fusion

Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3

Signaling through the primary cilium

© 2018 Wheway, Nazlamova and Hancock. The presence of single, non-motile "primary" cilia on the surface of epithelial cells has been well described since the 1960s. However, for decades these organelles were believed to be vestigial, with no remaining function, having lost their motility. It wasn't until 2003, with the discovery that proteins responsible for transport along the primary cilium are essential for hedgehog signaling in mice, that the fundamental importance of primary cilia in signal transduction was realized. Little more than a decade later, it is now clear that the vast majority of signaling pathways in vertebrates function through the primary cilium. This has led to the adoption of the term "the cells's antenna" as a description for the primary cilium. Primary cilia are particularly important during development, playing fundamental roles in embryonic patterning and organogenesis, with a suite of inherited developmental disorders known as the "ciliopathies" resulting from mutations in genes encoding cilia proteins. This review summarizes our current understanding of the role of these fascinating organelles in a wide range of signaling pathways

The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies

Nephronophthisis

Nephronophthisis (NPH) is an autosomal recessive disease characterized by a chronic tubulointerstitial nephritis that progress to terminal renal failure during the second decade (juvenile form) or before the age of 5 years (infantile form). In the juvenile form, a urine concentration defect starts during the first decade, and a progressive deterioration of renal function is observed in the following years. Kidney size may be normal, but loss of corticomedullary differentiation is often observed, and cysts occur usually after patients have progressed to end-stage renal failure. Histologic lesions are characterized by tubular basement membrane anomalies, tubular atrophy, and interstitial fibrosis. The infantile form is characterized by cortical microcysts and progression to end-stage renal failure before 5 years of age. Some children present with extrarenal symptoms: retinitis pigmentosa (Senior-Løken syndrome), mental retardation, cerebellar ataxia, bone anomalies, or liver fibrosis. Positional cloning and candidate gene approaches led to the identification of eight causative genes (NPHP1, 3, 4, 5, 6, 7, 8, and 9) responsible for the juvenile NPH and one gene NPHP2 for the infantile form. NPH and associated disorders are considered as ciliopathies, as all NPHP gene products are expressed in the primary cilia, similarly to the polycystic kidney disease (PKD) proteins

Mouse hitchhiker mutants have spina bifida, dorso-ventral patterning defects and polydactyly: identification of Tulp3 as a novel negative regulator of the Sonic hedgehog pathway

The mammalian Sonic hedgehog (Shh) signalling pathway is essential for embryonic development and the patterning of multiple organs. Disruption or activation of Shh signalling leads to multiple birth defects, including holoprosencephaly, neural tube defects and polydactyly, and in adults results in tumours of the skin or central nervous system. Genetic approaches with model organisms continue to identify novel components of the pathway, including key molecules that function as positive or negative regulators of Shh signalling. Data presented here define Tulp3 as a novel negative regulator of the Shh pathway. We have identified a new mouse mutant that is a strongly hypomorphic allele of Tulp3 and which exhibits expansion of ventral markers in the caudal spinal cord, as well as neural tube defects and preaxial polydactyly, consistent with increased Shh signalling. We demonstrate that Tulp3 acts genetically downstream of Shh and Smoothened (Smo) in neural tube patterning and exhibits a genetic interaction with Gli3 in limb development. We show that Tulp3 does not appear to alter expression or processing of Gli3, and we demonstrate that transcriptional regulation of other negative regulators (Rab23, Fkbp8, Thm1, Sufu and PKA) is not affected. We discuss the possible mechanism of action of Tulp3 in Shh-mediated signalling in light of these new data