Peter Kosa

Epithelial Integrity Is Maintained by a Matriptase-Dependent Proteolytic Pathway

A pericellular proteolytic pathway initiated by the transmembrane serine protease matriptase plays a critical role in the terminal differentiation of epidermal tissues. Matriptase is constitutively expressed in multiple other epithelia, suggesting a putative role of this membrane serine protease in general epithelial homeostasis. Here we generated mice with conditional deletion of the St14 gene, encoding matriptase, and show that matriptase indeed is essential for the maintenance of multiple types of epithelia in the mouse. Thus, embryonic or postnatal ablation of St14 in epithelial tissues of diverse origin and function caused severe organ dysfunction, which was often associated with increased permeability, loss of tight junction function, mislocation of tight junction-associated proteins, and generalized epithelial demise. The study reveals that the homeostasis of multiple simple and stratified epithelia is matriptase-dependent, and provides an important animal model for the exploration of this membrane serine protease in a range of physiological and pathological processes.

Regulation of cell surface protease matriptase by HAI2 is essential for placental development, neural tube closure and embryonic survival in mice

Hypomorphic mutations in the human SPINT2 gene cause a broad spectrum of abnormalities in organogenesis, including organ and digit duplications, atresia, fistulas, hypertelorism, cleft palate and hamartoma. SPINT2 encodes the transmembrane serine protease inhibitor HAI2 (placental bikunin), and the severe developmental effects of decreased HAI2 activity can be hypothesized to be a consequence of excess pericellular proteolytic activity. Indeed, we show here that HAI2 is a potent regulator of protease-guided cellular responses, including motogenic activity and transepithelial resistance of epithelial monolayers. Furthermore, we show that inhibition of the transmembrane serine protease matriptase (encoded by St14) is an essential function of HAI2 during tissue morphogenesis. Genetic inactivation of the mouse Spint2 gene led to defects in neural tube closure, abnormal placental labyrinth development associated with loss of epithelial cell polarity, and embryonic demise. Developmental defects observed in HAI2-deficient mice were caused by unregulated matriptase activity, as both placental development and embryonic survival in HAI2-deficient embryos were completely restored by the simultaneous genetic inactivation of matriptase. However, neural tube defects were detected in HAI2-deficient mice even in the absence of matriptase, although at lower frequency, indicating that the inhibition of additional serine protease(s) by HAI2 is required to complete neural development. Finally, by genetic complementation analysis, we uncovered a unique and complex functional interaction between HAI2 and the related HAI1 in the regulation of matriptase activity during development. This study indicates that unregulated matriptase-dependent cell surface proteolysis can cause a diverse array of abnormalities in mammalian development.

c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase.

The progression and negative outcome of a variety of human carcinomas are intimately associated with aberrant activity of the c-Met oncogene. The underlying cause of this dysregulation, however, remains a subject of discussion, as the majority of cancer patients do not present with activating mutations in c-Met receptor itself. In this study, we show that the oncogenic protease matriptase is ubiquitously co-expressed with the c-Met in human squamous cell carcinomas and amplifies migratory and proliferative responses of primary epithelial cells to the cognate ligand for c-Met, pro-hepatocyte growth factor/scatter factor (proHGF/SF), through c-Met and Gab1 signaling. Furthermore, the selective genetic ablation of c-Met from matriptase-expressing keratinocytes completely negates the oncogenic potential of matriptase. In addition, matriptase-dependent carcinoma formation could be blocked by the pharmacological inhibition of the Akt–mammalian target of Rapamycin (mTor) pathway. Our data identify matriptase as an initiator of c-Met-Akt–mTor-dependent signaling axis in tumors and reveal mTor activation as an essential component of matriptase/c-Met-induced carcinogenesis. The study provides a specific example of how epithelial transformation can be promoted by epigenetic acquisition of the capacity to convert a widely available paracrine growth factor precursor to its signaling competent state.

Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency–Associated Developmental Defects by Preventing Matriptase Activation

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1–deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2–deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.

Cerebrospinal fluid markers reveal intrathecal inflammation in progressive multiple sclerosis.

The management of complex patients with neuroimmunological diseases is hindered by an inability to reliably measure intrathecal inflammation. Currently implemented laboratory tests developed >40 years ago either are not dynamic or fail to capture low levels of central nervous system (CNS) inflammation. Therefore, we aimed to identify and validate biomarkers of CNS inflammation in 2 blinded, prospectively acquired cohorts of untreated patients with neuroimmunological diseases and embedded controls, with the ultimate goal of developing clinically useful tools.

Loss of Matriptase Suppression Underlies Spint1 Mutation-Associated Ichthyosis and Postnatal Lethality

Hepatocyte growth factor activator inhibitor-1 (HAI)-1 is an epithelial Kunitz-type transmembrane serine protease inhibitor that is encoded by the SPINT1 gene. HAI-1 displays potent inhibitory activity toward a large number of trypsin-like serine proteases. HAI-1 was recently shown to play an essential role in postnatal epithelial homeostasis. Thus, Spint1-deficient mice were found to display severe growth retardation and are unable to survive beyond postnatal day 16. The mice present histologically with overt hyperkeratosis of the forestomach, hyperkeratosis and acanthosis of the epidermis, and hypotrichosis associated with abnormal cuticle development. In this study, we show that loss of inhibition of a proteolytic pathway that is dependent on the type II transmembrane serine protease, matriptase, underlies the detrimental effects of postnatal Spint1 deficiency. Matriptase and HAI-1 precisely co-localize in all tissues that are affected by the Spint1 disruption. Spint1-deficient mice that have low matriptase levels, caused by a hypomorphic mutation in the St14 gene that encodes matriptase, not only survived the neonatal period but were healthy and displayed normal long-term survival. Furthermore, a detailed histological analysis of neonatal, young adult, as well as aged mice did not reveal any abnormalities in Spint1-deficent mice that have low matriptase levels. This study identifies matriptase suppression as an essential function of HAI-1 in postnatal tissue homeostasis.

Paradoxical Immune Responses in Non-HIV Cryptococcal Meningitis

The fungus Cryptococcus is a major cause of meningoencephalitis in HIV-infected as well as HIV-uninfected individuals with mortalities in developed countries of 20% and 30%, respectively. In HIV-related disease, defects in T-cell immunity are paramount, whereas there is little understanding of mechanisms of susceptibility in non-HIV related disease, especially that occurring in previously healthy adults. The present description is the first detailed immunological study of non-HIV-infected patients including those with severe central nervous system (s-CNS) disease to 1) identify mechanisms of susceptibility as well as 2) understand mechanisms underlying severe disease. Despite the expectation that, as in HIV, T-cell immunity would be deficient in such patients, cerebrospinal fluid (CSF) immunophenotyping, T-cell activation studies, soluble cytokine mapping and tissue cellular phenotyping demonstrated that patients with s-CNS disease had effective microbiological control, but displayed strong intrathecal expansion and activation of cells of both the innate and adaptive immunity including HLA-DR+ CD4+ and CD8+ cells and NK cells. These expanded CSF T cells were enriched for cryptococcal-antigen specific CD4+ cells and expressed high levels of IFN-γ as well as a lack of elevated CSF levels of typical T-cell specific Th2 cytokines -- IL-4 and IL-13. This inflammatory response was accompanied by elevated levels of CSF NFL, a marker of axonal damage, consistent with ongoing neurological damage. However, while tissue macrophage recruitment to the site of infection was intact, polarization studies of brain biopsy and autopsy specimens demonstrated an M2 macrophage polarization and poor phagocytosis of fungal cells. These studies thus expand the paradigm for cryptococcal disease susceptibility to include a prominent role for macrophage activation defects and suggest a spectrum of disease whereby severe neurological disease is characterized by immune-mediated host cell damage.

Insufficient disease inhibition by intrathecal rituximab in progressive multiple sclerosis

Inaccessibility of the inflammation compartmentalized to the central nervous system (CNS) may underlie the lack of efficacy of immunomodulatory treatments in progressive multiple sclerosis (MS). The double blind combination of Rituximab by IntraVenous and IntraThecAl injection versus placebo in patients with Low‐Inflammatory SEcondary progressive MS (RIVITALISE; NCT01212094) trial was designed to answer: (1) Whether an induction dose of intravenous and intrathecal rituximab efficiently depletes CNS B cells? and (2) If so, whether this leads to global inhibition of CNS inflammation and slowing of CNS tissue destruction?

Development of a Sensitive Outcome for Economical Drug Screening for Progressive Multiple Sclerosis Treatment

Therapeutic advance in progressive multiple sclerosis (MS) has been very slow. Based on the transformative role magnetic resonance imaging (MRI) contrast-enhancing lesions had on drug development for relapsing-remitting MS, we consider the lack of sensitive outcomes to be the greatest barrier for developing new treatments for progressive MS. The purpose of this study was to compare 58 prospectively acquired candidate outcomes in the real-world situation of progressive MS trials to select and validate the best-performing outcome. The 1-year pre-treatment period of adaptively designed IPPoMS (ClinicalTrials.gov #NCT00950248) and RIVITaLISe (ClinicalTrials.gov #NCT01212094) Phase II trials served to determine the primary outcome for the subsequent blinded treatment phase by comparing 8 clinical, 1 electrophysiological, 1 optical coherence tomography, 7 MRI volumetric, 9 quantitative T1 MRI, and 32 diffusion tensor imaging MRI outcomes. Fifteen outcomes demonstrated significant progression over 1 year (Δ) in the predetermined analysis and seven out of these were validated in two independent cohorts. Validated MRI outcomes had limited correlations with clinical scales, relatively poor signal-to-noise ratios (SNR) and recorded overlapping values between healthy subjects and MS patients with moderate-severe disability. Clinical measures correlated better, even though each reflects a somewhat different disability domain. Therefore, using machine-learning techniques, we developed a combinatorial weight-adjusted disability score (CombiWISE) that integrates four clinical scales: expanded disability status scale (EDSS), Scripps neurological rating scale, 25 foot walk and 9 hole peg test. CombiWISE outperformed all clinical scales (Δ = 9.10%; p = 0.0003) and all MRI outcomes. CombiWISE recorded no overlapping values between healthy subjects and disabled MS patients, had high SNR, and predicted changes in EDSS in a longitudinal assessment of 98 progressive MS patients and in a cross-sectional cohort of 303 untreated subjects. One point change in EDSS corresponds on average to 7.50 point change in CombiWISE with a standard error of 0.10. The novel validated clinical outcome, CombiWISE, outperforms the current broadly utilized MRI brain atrophy outcome and more than doubles sensitivity in detecting clinical deterioration in progressive MS in comparison to the scale traditionally used for regulatory approval, EDSS.

Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival.

The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival.