A PDZ Protein GIPC3 Positively Modulates Hedgehog Signaling and Melanoma Growth.

The hedgehog (Hh) pathway is essential for animal development, but aberrant activation promotes cancer growth. In this study, we show that GIPC3, a PDZ domain-containing protein with putative adaptor protein function, positively modulates Hh target gene expression in normal fibroblasts and melanoma cells and supports melanoma tumor growth. Using overexpression and epistasis studies, we show that Gipc3 potentiates Hh transcriptional output and that it modulates GLI-dependent transcription independently of Sufu. Whereas we find that GIPC3 protein does not interact with Hh pathway components, Ingenuity Pathway Analyses of GIPC3-interacting proteins identified by coimmunoprecipitation and mass spectrometry show an association with cancer pathogenesis. Subsequent interrogation of The Cancer Genome Atlas and the Human Protein Atlas databases reveals GIPC3 upregulation in many cancers. Using expression screens in selected groups of GIPC3-upregulated cancers with reported Hh pathway activation, we find a significant positive correlation of GIPC3 expression with Hh pathway components GLI1, GLI2, and GPR161 in melanoma lines. Consistently, GIPC3 knockdown in melanoma lines significantly reduces GLI1 and GLI2 expression, cell viability, colony formation, and allograft tumor growth. Our findings highlight previously unidentified roles of GIPC3 in potentiating Hh response and melanoma tumorigenesis and suggest that GIPC3 modulation on Hh signaling may be targeted to reduce melanoma growth.

LMBD1 protein participates in cell mitosis by regulating microtubule assembly.

LMBD1 was previously demonstrated to regulate the endocytosis of insulin receptor on the cell surface and to mediate the export of cobalamin from the lysosomes to the cytosol, but little is known about its function in mitosis. In this study, interactome analysis data indicate that LMBD1 is involved in cytoskeleton regulation. Both immunoprecipitation and GST pulldown assays demonstrated the association of LMBD1 with tubulin. Immunofluorescence staining also showed the colocalization of LMBD1 with microtubule in both interphase and mitotic cells. LMBD1 specifically accelerates microtubule assembly dynamics in vitro and antagonizes the microtubule-disruptive effect of vinblastine. In addition, LMBRD1-knockdown impairs mitotic spindle formation, inhibits tubulin polymerization, and diminishes the mitosis-associated tubulin acetylation. The reduced acetylation can be reversed by ectopic expression of LMBD1 protein. These results suggest that LMBD1 protein stabilizes microtubule intermediates. Furthermore, embryonic fibroblasts derived from Lmbrd1 heterozygous knockout mice showed abnormality in microtubule formation, mitosis, and cell growth. Taken together, LMBD1 plays a pivotal role in regulating microtubule assembly that is essential for the process of cell mitosis.

Differential activation behavior of dermal dendritic cells underlies the strain-specific Th1 responses to single epicutaneous immunization.

BACKGROUND: Epicutaneous immunization with allergens is an important sensitization route for atopic dermatitis. We recently showed in addition to the Th2 response following single epicutaneous immunization, a remarkable Th1 response is induced in B6 mice, but not in BALB/c mice, mimicking the immune response to allergens in human non-atopics and atopics. OBJECTIVE: We investigated the underlying mechanisms driving this differential Th1 response between BALB/c and B6 mice. METHODS: We characterized dermal dendritic cells by flow cytometric analysis. We measured the induced Th1/Th2 responses by measuring the IFN-γ/IL-13 contents of supernatants of antigen reactivation cultures of lymph node cells. RESULTS: We demonstrate that more dermal dendritic cells with higher activation status migrate into draining lymph nodes of B6 mice compared to BALB/c mice. Dermal dendritic cells of B6 mice have a greater ability to capture protein antigen than those of BALB/c mice. Moreover, increasing the activation status or amount of captured antigen in dermal dendritic cells induced a Th1 response in BALB/c mice. Further, differential activation behavior, but not antigen-capturing ability of dermal dendritic cells between BALB/c and B6 mice is dendritic cell-intrinsic. CONCLUSION: These results show that the differential activation behavior of dermal dendritic cells underlies the strain-specific Th1 responses following single epicutaneous immunization. Furthermore, our findings highlight the potential differences between human atopics and non-atopics and provide useful information for the prediction and prevention of atopic diseases.

Suppressive effects of primed eosinophils on single epicutaneous sensitization through regulation of dermal dendritic cells.

Eosinophils are multifunctional innate immune cells involved in many aspects of innate and adaptive immunity. Epicutaneous sensitization with protein allergen is an important sensitization route for atopic dermatitis. In this study, using a murine single protein-patch model, we show that eosinophils of a primed status accumulate in draining lymph nodes following single epicutaneous sensitization. Further, depletion of eosinophils results in enhancement of the induced Th1/Th2 immune responses, whereas IL-5-induced hypereosinophilia suppresses these responses. Mechanistically, primed eosinophils cause a reduction in the numbers and activation status of dermal dendritic cells in draining lymph nodes. Collectively, these results demonstrate that primed eosinophils exert suppressive effects on single epicutaneous sensitization through regulation of dermal dendritic cells. Thus, these findings highlight the critical roles of eosinophils in the pathogenesis of atopic dermatitis with important clinical implications for the prevention of allergen sensitization.

Dermal dendritic cells, but not Langerhans cells, are critical in murine single epicutaneous sensitization.

A murine repeated protein-patch model has been established to study epicutaneous sensitization in atopic dermatitis. This model has shown a predominant Th2 and a weak Th1 response in both BALB/c and C57BL/6 mice. However, Th responses induced in the repeated model are not consistent with the generally accepted theory that BALB/c and C57BL/6 mice are Th2 and Th1 prone and are representatives of human atopy and non-atopy, respectively. In this study, a single protein-patch model was established, which showed in addition to the Th2 response, a remarkable Th1 response in C57BL/6 mice, but not in BALB/c mice. Moreover, using muLangerin-DTR mice, we demonstrated that dermal dendritic cells, but not Langerhans cells, are critical in single epicutaneous sensitization in both strains of mice.