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Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells. This observation is significant because InsP8 is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP8 within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP8 levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP6 and 5-InsP7, are very similar in both HCT116NIH and HCT116UCL lines. We also developed a new HPLC procedure to record 1-InsP7 levels directly (for the first time in any mammalian cell line); 1-InsP7 comprised <2% of total InsP7 in HCT116NIH and HCT116UCL lines. The elevated levels of InsP8 in the HCT116UCL lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP8. We discuss how the divergent PP-InsP profiles of the newly-designated HCT116NIH and HCT116UCL lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP8 turnover, and may also yield new directions for studying InsP8 function. [ABSTRACT FROM AUTHOR]

Inhalational anthrax is initiated by pulmonary exposure to Bacillus anthracis spores. Spore entry into lung epithelial cells is observed both in vitro and in vivo and evidence suggests it is important for bacterial dissemination and virulence. However the specific host receptor and spore factor that mediate the entry process were unknown. Here, we report that integrin α2β1 is a major receptor for spore entry. This is supported by results from blocking antibodies, siRNA knock-down, colocalization, and comparison of spore entry into cells that do or do not express α2. BclA, a major spore surface protein, is found to be essential for entry and α2β1-mediated entry is dependent on BclA. However, BclA does not appear to bind directly to α2. Furthermore, spore entry into α2-expressing cells is dramatically reduced in the absence of serum, suggesting that additional factors are involved. Finally, complement component C1q, also an α2β1 ligand, appears to act as a bridging molecule or a cofactor for BclA/α2β1-mediated spore entry and BclA binds to C1q in a dose-dependent and saturable manner. These findings suggest a novel mechanism for pathogen entry into host cells as well as a new function for C1q-integrin interactions. The implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

The colon epithelium undergoes constant self-renewal in order to maintain its function as a nutrient sensor and a physical barrier from the commensal microbiota. Colon epithelium homeostasis is altered by inflammation and tissue injury/repair. In excess of which, contributes to the development of colon cancer. Here, we investigated the impact of IL-17A on the colon epithelium homeostasis and tumor development. IL-17A signals through a heterodimeric receptor complex composed of IL-17RA and IL-17RC. We found that the colon epithelium from IL-17RC-deficient mice displayed reduced proliferation, increased cell apoptosis and increased permeability after DSS challenge compared to control mice. Moreover, the IL-17RC-deficient mice developed significantly fewer colonic tumors compared to control mice in the AOM/DSS model of colitis-associated cancer. We then aimed to identify the cell-type responsible for IL-17A-mediated impact on homeostasis and tumorigenesis. Presumably, this IL-17-responsive cell population would sustain the initial mutation induced by carcinogen AOM, self replicate, and ultimately give rise to the neoplastic foci. Since the renewal of the colon epithelium relies upon a stem-cell niche (LGR5-positive cells) at the base of each crypt, we specifically ablated IL-17 signaling by deleting the IL-17R adaptor protein Act1 from this compartment. Following the AOM/DSS regimen the Act1CSC-KO mice developed significantly fewer colonic tumors compared to littermates. Mechanistically, IL-17A induced the activation of ERK5, ERK1/2 and JNK in colon epithelial cells. The activation of ERK5 and ERK1/2 were diminished in colon tissues of IL-17RC-deficient mice compared to that in the control mice with or without DSS treatment, implicating these MAPKs in IL-17R-dependent colon epithelium homeostasis. Furthermore, this pathway relied upon IL-17RC-dependent Act1 recruitment of TRAF4. We propose that IL-17R-Act1-TRAF4 cascade activates MAPKs in the LGR5-positive stem cells to promote regeneration after injury, consequently contributing to colon tumorigenesis by sustaining stem cells harboring a critical genetic lesion. [ABSTRACT FROM AUTHOR]

Technical challenges have to date prevented a complete profiling of the levels of myo -inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP 5 and 2-PP-InsP 5 . The latter has not previously been described in nature. The analysis of feces and the commercial mouse diet suggests that the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP 5 and 2-PP-InsP 5 . In summary, our study substantially expands PP-InsP biology in mammals., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Lrig1 marks a distinct population of stem cells restricted to the upper pilosebaceous unit in normal epidermis. Here we report that IL-17A-mediated activation of EGFR plays a critical role in the expansion and migration of Lrig1 + stem cells and their progenies in response to wounding, thereby promoting wound healing and skin tumorigenesis. Lrig1-specific deletion of the IL-17R adaptor Act1 or EGFR in mice impairs wound healing and reduces tumor formation. Mechanistically, IL-17R recruits EGFR for IL-17A-mediated signaling in Lrig1 + stem cells. While TRAF4, enriched in Lrig1 + stem cells, tethers IL-17RA and EGFR, Act1 recruits c-Src for IL-17A-induced EGFR transactivation and downstream activation of ERK5, which promotes the expansion and migration of Lrig1 + stem cells. This study demonstrates that IL-17A activates the IL-17R-EGFR axis in Lrig1 + stem cells linking wound healing to tumorigenesis., (© 2018 Chen et al.)