Mutations in LRPAP1 are associated with severe myopia in humans.

Myopia is an extremely common eye disorder but the pathogenesis of its isolated form, which accounts for the overwhelming majority of cases, remains poorly understood. There is strong evidence for genetic predisposition to myopia, but determining myopia genetic risk factors has been difficult to achieve. We have identified Mendelian forms of myopia in four consanguineous families and implemented exome/autozygome analysis to identify homozygous truncating variants in LRPAP1 and CTSH as the likely causal mutations. LRPAP1 encodes a chaperone of LRP1, which is known to influence TGF-ß activity. Interestingly, we observed marked deficiency of LRP1 and upregulation of TGF-ß in cells from affected individuals, the latter being consistent with available data on the role of TGF-ß in the remodeling of the sclera in myopia and the high frequency of myopia in individuals with Marfan syndrome who characteristically have upregulation of TGF-ß signaling. CTSH, on the other hand, encodes a protease and we show that deficiency of the murine ortholog results in markedly abnormal globes consistent with the observed human phenotype. Our data highlight a role for LRPAP1 and CTSH in myopia genetics and demonstrate the power of Mendelian forms in illuminating new molecular mechanisms that may be relevant to common phenotypes.

The activity and localization patterns of cathepsins B and X in cells of the mouse gastrointestinal tract differ along its length.

Cysteine cathepsins are expressed in most tissues, including the gastrointestinal tract. We demonstrated an involvement of mouse intestinal cathepsin B in extracellular matrix remodeling for regeneration from trauma. The present study aimed at elucidating roles of cysteine cathepsins in the non-traumatized gastrointestinal tract of mice. Thus we investigated expression and localization patterns of cathepsin B and its closest relative, cathepsin X, along the length of the gastrointestinal tract, and determined the effects of their absence. Cathepsin B showed the highest protein levels in the anterior segments of the gastrointestinal tract, whereas the highest activity was observed in the jejunum, as revealed by cathepsin B-specific activity-based probe labeling. Cathepsin X was most abundant in the jejunum and protein levels were elevated in duodenum and colon of Ctsb-/- mice. The segmental pattern of cathepsin expression was reflected by a compartmentalized distribution of junction proteins and basal lamina constituents, changes in tissue architecture and altered activities of the brush border enzyme aminopeptidase N. In conclusion, we observed different compensatory effects and activity levels of cysteine peptidases along the length of the small and large intestines in a segment-specific manner suggesting specific in situ functions of these enzymes in particular parts of the gastrointestinal tract.

Unimpeded skin carcinogenesis in K14-HPV16 transgenic mice deficient for plasminogen activator inhibitor.

Angiogenesis, extracellular matrix remodeling and cell migration are associated with cancer progression and involve at least, the plasminogen activating system and its main physiological inhibitor, the plasminogen activator inhibitor-1 (PAI-1). Considering the recognized importance of PAI-1 in the regulation of tumor angiogenesis and invasion in murine models of skin tumor transplantation, we explored the functional significance of PAI-1 during early stages of neoplastic progression in the transgenic mouse model of multistage epithelial carcinogenesis (K14-HPV16 mice). We have studied the effect of genetic deletion of PAI-1 on inflammation, angiogenesis, lymphangiogenesis and tumor progression. In this model, PAI-1 deficiency neither impaired keratinocyte hyperproliferation or tumor development nor affected the infiltration of inflammatory cells and development of angiogenic or lymphangiogenic vasculature. We are reporting evidence for concomitant lymphangiogenic and angiogenic switches independent to PAI-1 status. Taken together, these data indicate that PAI-1 is not rate limiting for neoplastic progression and vascularization during premalignant progression, or that there is a functional redundancy between PAI-1 and other tumor regulators, masking the effect of PAI-1 deficiency in this long-term model of multistage epithelial carcinogenesis.

Cellular senescence induced by cathepsin X downregulation.

Cellular senescence represents a powerful tumor suppressor mechanism to prevent proliferation and invasion of malignant cells. Since tumor cells as well as primary fibroblasts lacking the lysosomal cysteine-type carboxypeptidase cathepsin X exhibit a reduced invasive capacity, we hypothesized that the underlying reason may be the induction of cellular senescence. To investigate the cellular and molecular mechanisms leading to diminished migration/invasion of cathepsin X-deficient cells, we have analyzed murine embryonic fibroblasts (MEF) derived from cathepsin X-deficient mice and neonatal human dermal fibroblasts (NHDF) transfected with siRNAs targeting cathepsin X. Remarkably, both cell types exhibited a flattened and enlarged cell body, a characteristic phenotype of senescent cells. Additional evidence for accelerated senescence was obtained by detection of the common senescence marker ß-galactosidase. Further examination revealed increased expression levels of senescence-associated genes such as p16, p21, p53, and caveolin in these cells along with a reduced proliferation rate. The accelerated cellular senescence induced by cathepsin X deficiency was rescued by simultaneous expression of exogenous cathepsin X. Finally, cell cycle analysis confirmed a marked reduction of the synthesis rate and prolongation of the S-phase, while susceptibility to apoptosis of cathepsin X-deficient cells remained unchanged. In conclusion, cathepsin X deficiency leads to accelerated cellular senescence and consequently to diminished cellular proliferation and migration/invasion implying a potential role of cathepsin X in bypassing cellular senescence.

The inherent differentiation program of short-term hematopoietic repopulating cells changes during human ontogeny.

Human umbilical cord blood (CB) could be an attractive source of hematopoietic repopulating cells for clinical stem cell therapy because of its accessibility and low propensity for unwanted immune reaction against the host. However, CB recipients suffer from severely delayed and often chronically deficient platelet recovery of unknown cause. Here we show that human short-term repopulating cells (STRCs), which predominantly carry early hematopoietic reconstitution after transplantation, display an intrinsically fixed differentiation program in vivo that changes during ontogeny. Compared to adult sources of hematopoietic cells, CB myeloidrestricted STRC-M showed a markedly reduced megakaryocytic and erythroid cell output in the quantitative xenotransplantation of human short-term hematopoiesis in NOD/SCID-beta2m(-/-) mice. This output in vivo was not altered by pre-treating CB cells before transplantation with growth factors that effectively stimulate megakaryocytopoiesis in vitro. Moreover, injecting mice with granulocyte colony-stimulating factor did not affect the differentiation of human STRC. These findings demonstrate that the differentiation capacity of human STRCs is developmentally regulated by mechanisms inaccessible to currently available hematopoietic growth factors, and explain why thrombopoiesis is deficient in clinical CB transplantation.

Anti-PSMA immunotoxin as novel treatment for prostate cancer? High and specific antitumor activity on human prostate xenograft tumors in SCID mice.

BACKGROUND: Expression of the prostate specific membrane antigen (PSMA) is highly restricted to prostate epithelial cells. Therefore, toxin-based immunotherapy against this antigen may represent an alternative therapeutic option for prostate cancer. For these purposes, the effects of the recombinant anti-PSMA immunotoxin A5-PE40 on prostate tumor growth were investigated in vitro and in vivo. METHODS: The in vitro binding and cytotoxicity of A5-PE40 were tested on the PSMA-expressing prostate cancer cell line C4-2 and on the PSMA-negative cell line DU145 by flow cytometry and WST assays. The binding of the immunotoxin to SCID mouse xenografts and to various mouse organs was examined by Western blot analysis. In vivo, the antitumor activity of the immunotoxin was tested by injecting A5-PE40 in mice bearing C4-2 or DU145 xenografts. RESULTS: In vitro, a specific binding of A5-PE40 to C4-2 cells could be shown with a concentration-dependent cytotoxicity (IC(50) value=220 pM). In the next step, a specific binding of the immunotoxin to C4-2 xenografts could be demonstrated. In contrast, no binding on mouse organs expressing high homologous mouse PSMA was found. The treatment of mice with C4-2 tumors caused a significant inhibition of tumor growth in vivo, whereas DU145 xenografts remained totally unaffected. CONCLUSIONS: A5-PE40 represents a recombinant anti-PSMA immunotoxin with potent antitumor activity in mice bearing human prostate cancer xenograft tumors. Therefore, A5-PE40 could be a promising candidate for therapeutic applications in patients with prostate cancer.