Generation of a tissue‐specific transgenic model for K8 phosphomutants: A tool to investigate the role of K8 phosphorylation during skin carcinogenesis in vivo

To inactivate chronically the β2-integrin CD11b (Mac-1), we made a transgenic model in mice in which we expressed the CD11b antagonist polypeptide neutrophil inhibitory factor (NIF). Using these mice, we determined the in vivo effects of CD11b inactivation on polymorphonuclear leukocyte (PMN) function and acute lung injury (ALI) induced by Escherichia coli septicemia. In wild-type PMNs, CD11b expression was induced within 1 h after E. coli challenge, whereas this response was significantly reduced in NIF+/+ PMNs. Coimmunoprecipitation studies showed that NIF associated with CD11b in NIF+/+ PMNs. To validate the effectiveness of CD11b blockade, we compared PMN function in NIF+/+ and Mac-1-deficient (Mac-1−/−) mice. Adhesion of both Mac-1−/− and NIF+/+ PMNs to endothelial cells in response to LPS was reduced in both types of PMNs and fully blocked only by the addition of anti-CD11a monoclonal antibody. This finding is indicative of intact CD11a function in the NIF+/+ PMNs but the blockade of CD11b function. CD11b inactivation in NIF+/+ mice interfered with lung PMN infiltration induced by E. coli and prevented the increase in lung microvessel permeability and edema formation, with most of the protection seen in the 1-h period after the E. coli. Thus our results demonstrate that CD11b plays a crucial role in mediating lung PMN sequestration and vascular injury in the early phase of gram-negative septicemia. The NIF+/+ mouse model, in which CD11b is inactivated by binding to NIF, is a potentially useful model for in vivo assessment of the role of PMN CD11b in the mechanism of vascular inflammation.

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Dependence of Mycobacterium bovis BCG on Anaerobic Nitrate Reductase for Persistence Is Tissue Specific

Infection and Immunity ◽

10.1128/iai.70.1.286-291.2002 ◽

2002 ◽

Vol 70 (1) ◽

pp. 286-291 ◽

Cited By ~ 72

Author(s):

Christian Fritz ◽

Silvia Maass ◽

Andreas Kreft ◽

Franz-Christoph Bange

Keyword(s):

Nitrate Reductase ◽

Mycobacterium Bovis ◽

Vaccine Development ◽

Nitrate Respiration ◽

Apparent Paradox ◽

Tissue Specific ◽

Deficient Mice

ABSTRACT Mycobacterium bovis BCG, the only presently available vaccine against tuberculosis, was obtained from virulent M. bovis after serial passages in vitro. The vaccine strain retained at least some of its original virulence, as it persists in immune-competent hosts and occasionally may cause fatal disease in immune-deficient hosts. Mycobacterial persistence in vivo is thought to depend on anaerobic metabolism, an apparent paradox since all mycobacteria are obligate aerobes. Here we report that M. bovis BCG lacking anaerobic nitrate reductase (NarGHJI), an enzyme essential for nitrate respiration, failed to persist in the lungs, liver, and kidneys of immune-competent (BALB/c) mice. In immune-deficient (SCID) mice, however, bacilli caused chronic infection despite disruption of narG, even if growth of the mutant was severely impaired in lungs, liver, and kidneys. Persistence and growth of BCG in the spleens of either mouse strain appeared largely unaffected by lack of anaerobic nitrate reductase, indicating that the role of the enzyme in pathogenesis is tissue specific. These data suggest first that anaerobic nitrate reduction is essential for metabolism of M. bovis BCG in immune-competent but not immune-deficient mice and second that its role in mycobacterial disease is tissue specific, both of which are observations with important implications for pathogenesis of mycobacteria and vaccine development.

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Redundant Role of Tissue-Selective TAFII105 in B Lymphocytes

Molecular and Cellular Biology ◽

10.1128/mcb.22.18.6564-6572.2002 ◽

2002 ◽

Vol 22 (18) ◽

pp. 6564-6572 ◽

Cited By ~ 8

Author(s):

Richard N. Freiman ◽

Shane R. Albright ◽

Leslie E. Chu ◽

Shuang Zheng ◽

Hong-Erh Liang ◽

...

Keyword(s):

B Cells ◽

Rna Polymerase Ii ◽

B Lymphocyte ◽

Critical Role ◽

Central Component ◽

Related Factors ◽

Tissue Specific ◽

Regulated Gene Expression

ABSTRACT Regulated gene expression is a complex process achieved through the function of multiple protein factors acting in concert at a given promoter. The transcription factor TFIID is a central component of the machinery regulating mRNA synthesis by RNA polymerase II. This large multiprotein complex is composed of the TATA box binding protein (TBP) and several TBP-associated factors (TAFIIs). The recent discovery of multiple TBP-related factors and tissue-specific TAFIIs suggests the existence of specialized TFIID complexes that likely play a critical role in regulating transcription in a gene- and tissue-specific manner. The tissue-selective factor TAFII105 was originally identified as a component of TFIID derived from a human B-cell line. In this report we demonstrate the specific induction of TAFII105 in cultured B cells in response to bacterial lipopolysaccharide (LPS). To examine the in vivo role of TAFII105, we have generated TAFII105-null mice by homologous recombination. Here we show that B-lymphocyte development is largely unaffected by the absence of TAFII105. TAFII105-null B cells can proliferate in response to LPS, produce relatively normal levels of resting antibodies, and can mount an immune response by producing antigen-specific antibodies in response to immunization. Taken together, we conclude that the function of TAFII105 in B cells is likely redundant with the function of other TAFII105-related cellular proteins.

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Muscle-specific overexpression of lipoprotein lipase in transgenic mice results in increased α-tocopherol levels in skeletal muscle

Biochemical Journal ◽

10.1042/bj3180015 ◽

1996 ◽

Vol 318 (1) ◽

pp. 15-19 ◽

Cited By ~ 56

Author(s):

Wolfgang SATTLER ◽

Sanja LEVAK-FRANK ◽

Herbert RADNER ◽

Gerhard M. KOSTNER ◽

Rudolf ZECHNER

Keyword(s):

Skeletal Muscle ◽

Adipose Tissue ◽

Transgenic Mice ◽

Cardiac Muscle ◽

Lipoprotein Lipase ◽

Excellent Correlation ◽

Peripheral Tissues ◽

Tissue Specific

Lipoprotein lipase (LPL) has been implicated in the delivery of chylomicron-located α-tocopherol (α-TocH) to peripheral tissues. To investigate the role of LPL in the cellular uptake of α-TocH in peripheral tissue in vivo, three lines of transgenic mice [mouse creatine kinase- (MCK) L, MCK-M and MCK-H] expressing various amounts of human LPL were compared with regard to α-TocH levels in plasma, skeletal muscle, cardiac muscle, adipose tissue and brain. Depending on the copy number of the transgene, LPL activity was increased 3- to 27-fold in skeletal muscle and 1.3- to 3.7-fold in cardiac muscle. The intracellular levels of α-TocH in skeletal muscle were significantly increased in MCK-M and MCK-H animals and correlated highly with the tissue-specific LPL activity (r = 0.998). The highest levels were observed in MCK-H (21.4 nmol/g) followed by MCK-M (13.3 nmol/g) and MCK-L (8.2 nmol/g) animals when compared with control mice (7.3 nmol/g). Excellent correlation was also observed between intracellular α-TocH and non-esterified fatty acid (NEFA) levels (r = 0.998). Although LPL activities in cardiac muscle were also increased in the transgenic mouse lines, α-TocH concentrations in the heart remained unchanged. Similarly, α-TocH levels in plasma, adipose tissue and brain were unaffected by the tissue specific overexpression of LPL in muscle. The transgenic model presented in this report provides evidence that the uptake of α-TocH in muscle is directly dependent on the level of LPL expression in vivo. Increased intracellular α-TocH concentrations with increased triglyceride lipolysis and NEFA uptake might protect the myocyte from oxidative damage during increased β-oxidation.

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Role of c-Met/β1 integrin complex in the metastatic cascade

10.1101/2020.04.20.051888 ◽

2020 ◽

Author(s):

Darryl Lau ◽

Harsh Wadhwa ◽

Sweta Sudhir ◽

Saket Jain ◽

Ankush Chandra ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Human Cancer ◽

Β1 Integrin ◽

Tissue Specific ◽

Metastatic Cascade ◽

Kinase C

ABSTRACTMetastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. While individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a structural complex between receptor tyrosine kinase c-Met and β1 integrin in metastases. Using novel cell culture and in vivo assays, we found that c-Met/β1 complex induction promotes breast cancer intravasation and adhesion to the vessel wall, but does not increase extravasation. These effects may be driven by the ability of the c-Met/β1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/β1 complex induction. A β1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/β1 binding, decreased invasion and mesenchymal gene expression and morphology of breast cancer cells. Bone-seeking breast cancer cells exhibited higher c-Met/β1 complex levels than parental controls and preferentially adhere to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/β1 levels than brain metastases. Thus, the c-Met/β1 complex drives breast cancer cell intravasation and preferential affinity for bone tissue-specific matrix. Pharmacological targeting of the complex may prevent metastases, particularly osseous metastases.

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Elucidation of new vitamin K function aiming at application to cardiovascular disease prevention and treatment

Impact ◽

10.21820/23987073.2021.5.37 ◽

2021 ◽

Vol 2021 (5) ◽

pp. 37-39

Author(s):

Kimie Nakagawa

Keyword(s):

Cardiovascular Disease ◽

Disease Prevention ◽

Vitamin K ◽

Human Body ◽

Physiological Function ◽

Specific Gene ◽

Tissue Specific ◽

Prevention And Treatment

Although Vitamin K is known to be required by the human body for blood clotting and bone metabolism, there remain many unknowns about this group of vitamins and there is a lack of biochemical research on vitamin K. Limitations in knowledge about vitamin k may mean that decisions about the vitamin's uptake and bone health may not be optimal. Professor Kimie Nakagawa Laboratory of Hygienic Sciences, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Japan, is performing detailed investigations on vitamin K to shed light on its specific functions in the human body and contribute to advancements in disease prevention and treatment, including cardiovascular disease. Her studies centre on UBIAD1 (UbiA prenyltransferase domain containing protein 1) and MK-4 (menaquinone-4). She is seeking to elucidate the role of MK-4 in the human body and reveal the unknown functions of UBIAD1. Nakagawa and the teams are doing so using UBIAD1 tissue-specific gene-deficient mice, which they are analysing in order to clarify the significance of biosynthesis of MK-4 in vivo and unravel the role of UBIAD1 and MK-4 in each tissue. Specific research goals for Nakagawa are to elucidate vitamin K conversion mechanisms, establish improved understanding of the physiological function of the vitamin, identify vitamin K converting enzymes and learn and explain more about the physiological function of this enzyme. The team made an important discovery in that UBIAD1 is the enzyme responsible for the conversion of vitamin k to MK-4 and the researchers will continue to build on this groundbreaking finding.

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CSIG-10. ROLE OF c-Met/β1 INTEGRIN COMPLEX IN THE ESTABLISHMENT OF BRAIN METASTASES

Neuro-Oncology ◽

10.1093/neuonc/noz175.181 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi46-vi46

Author(s):

Harsh Wadhwa ◽

Darryl Lau ◽

Ankush Chandra ◽

Alan Nguyen ◽

Sumedh Shah ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cell Population ◽

Proximity Ligation Assay ◽

Breast Adenocarcinoma ◽

Β1 Integrin ◽

Primary Tumors ◽

Tissue Specific ◽

Metastatic Cascade

Abstract INTRODUCTION Metastases cause 90% of human cancer deaths. The metastatic cascade involves five steps: invasion, intravasation, extravasation, colonization, and proliferation. While individual mediators of these processes have been investigated, their interactions remain undefined. We previously demonstrated increased formation of a structural complex between receptor tyrosine kinase c-Met and β1 integrin in metastases compared to primary tumors. We used novel cell culture models and in vivo assays to define the role of this complex in individual steps of the metastatic cascade. METHODS The iDimerize heterodimer system was inserted into MDA-MB-231 breast adenocarcinoma cells, allowing c-Met/β1 heterodimerization induction via A/C heterodimerizer treatment. Scratch assays and novel transwell assay modifications were used to measure migration, invasion, intravasation, and extravasation. Proximity ligation assay was performed to measure c-Met/β1 complex. Nanostring panel was used to transcriptionally profile cells. RESULTS c-Met/β1 complex induction promotes breast cancer invasion (p< 0.001), migration (p< 0.05), intravasation (p< 0.01), and adhesion to the vessel wall (p< 0.01). However, it does not increase extravasation in culture or in vivo. These effects may be driven by the ability of c-Met/β1 to increase mesenchymal character (p< 0.05) and stem cell population (p< 0.001). Nanostring analysis revealed upregulated Wnt and hedgehog pathways after c-Met/β1 complex induction, particularly WNT7B (p< 0.05). OS2966, an antibody preventing c-Met/β1 binding, decreased invasion (p< 0.05), intravasation (p< 0.05), and mesenchymal morphology (p< 0.001) and gene expression (p< 0.001). Brain- and bone-seeking breast cancer cells have higher c-Met/β1 complex than controls (p< 0.05) and preferentially adhere to tissue-specific matrix (p< 0.01). CONCLUSIONS The c-Met/β1 complex drives breast cancer cell intravasation. While extravasation is not affected by the complex, preferential affinity for tissue-specific matrix enables the c-Met/β1 complex to drive breast cancer metastases to brain and bone. Pharmacological targeting of the complex may prevent metastases, particularly to the brain and bone.

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Analysis of the role of Nidogen/entactin in basement membrane assembly and morphogenesis in Drosophila

10.1101/346270 ◽

2018 ◽

Author(s):

Jianli Dai ◽

Beatriz Estrada ◽

Sofie Jacobs ◽

Besaiz J. Sánchez-Sánchez ◽

Jia Tang ◽

...

Keyword(s):

Adipose Tissue ◽

Type Iv Collagen ◽

Collagen Iv ◽

Basement Membranes ◽

Extracellular Matrices ◽

Tissue Specific ◽

Type Iv

AbstractBasement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen interlinked by the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg on morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from in vitro and cultured embryonic tissues experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival, strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.Author SummaryBasement membranes (BMs) are thin layers of specialized extracellular matrices present in every tissue of the human body. Its main constituents are two networks of Laminin and Type IV Collagen linked by Nidogen (Ndg) and proteoglycans. They form an organized scaffold that regulates organ morphogenesis and function. Mutations affecting BM components are associated with organ dysfunction and several congenital diseases. Thus, a better comprehension of BM assembly and maintenance will not only help to learn more about organogenesis but also to a better understanding and, hopefully, treatment of these diseases. Here, we have used Drosophila to analyse the role of Ndg in BM formation in vivo. Elimination of Ndg in worms and mice does not affect survival, strongly questioning its proposed linking role, derived from in vitro experiments. Here, we show that in the fly Ndg is dispensable for BM assembly and preservation in many tissues, but absolutely required in others. Furthermore, our functional study of the different Ndg domains challenges the significance of some interactions between BM components derived from in vitro experiments, while confirming others, and reveals a new key requirement for the Rod domain in Ndg function and incorporation into BMs.

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Tolerogenic Dendritic Cell-Based Approaches in Autoimmunity

International Journal of Molecular Sciences ◽

10.3390/ijms22168415 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8415

Author(s):

Laura Passeri ◽

Fortunato Marta ◽

Virginia Bassi ◽

Silvia Gregori

Keyword(s):

T Cells ◽

Autoimmune Diseases ◽

Organ Damage ◽

Dc Functions ◽

Tissue Specific ◽

Research Areas ◽

Tolerogenic Dendritic Cell

Dendritic cells (DCs) dictate the outcomes of tissue-specific immune responses. In the context of autoimmune diseases, DCs instruct T cells to respond to antigens (Ags), including self-Ags, leading to organ damage, or to becoming regulatory T cells (Tregs) promoting and perpetuating immune tolerance. DCs can acquire tolerogenic properties in vitro and in vivo in response to several stimuli, a feature that opens the possibility to generate or to target DCs to restore tolerance in autoimmune settings. We present an overview of the different subsets of human DCs and of the regulatory mechanisms associated with tolerogenic (tol)DC functions. We review the role of DCs in the induction of tissue-specific autoimmunity and the current approaches exploiting tolDC-based therapies or targeting DCs in vivo for the treatment of autoimmune diseases. Finally, we discuss limitations and propose future investigations for improving the knowledge on tolDCs for future clinical assessment to revert and prevent autoimmunity. The continuous expansion of tolDC research areas will lead to improving the understanding of the role that DCs play in the development and treatment of autoimmunity.

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CSIG-04. ROLE OF c-Met/β1 INTEGRIN COMPLEX IN THE METASTATIC CASCADE

Neuro-Oncology ◽

10.1093/neuonc/noaa215.116 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii28-ii28

Author(s):

Sweta Sudhir ◽

Darryl Lau ◽

Harsh Wadhwa ◽

Saket Jain ◽

Ankush Chandra ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Human Cancer ◽

Β1 Integrin ◽

Blocking Antibody ◽

Tissue Specific ◽

Metastatic Cascade

Abstract Metastases cause 90% of human cancer deaths. The metastatic cascade involves 5 steps: local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. While individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a structural complex between receptor tyrosine kinase c-Met and β1 integrin in metastases that form under certain biological and therapeutic inducers, including bevacizumab. Using novel cell culture and in vivo assays, we found that c-Met/β1 complex induction promotes breast cancer intravasation and adhesion to the vessel wall but does not increase extravasation. These effects may be driven by the ability of the c-Met/β1 complex to increase mesenchymal and stem cell characteristics in breast cancer cells. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/β1 complex induction in breast cancer cells. We subsequently used CRISPR to introduce a β1 integrin point mutation that prevented binding to c-Met and led to reduced intravasation, confirming the importance of c-Met/β1 integrin binding for the metastatic cascade. OS2966, a therapeutic B1 integrin blocking antibody, disrupted c-Met/β1 binding as well, and decreased invasion, mesenchymal gene expression, and mesenchymal morphology of breast cancer cells. Bone-seeking breast cancer cells exhibited higher c-Met/β1 complex levels than parental controls and preferentially adhere to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/β1 complex levels than brain metastases. Thus, our research suggests the c-Met/β1 complex drives breast cancer cell intravasation and preferential affinity for bone tissue-specific matrix. Pharmacological targeting of the complex may prevent metastases, particularly osseous metastases.

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