1994 — 1998 |
Kapila, Yvonne L |
K15Activity Code Description: Undocumented code - click on the grant title for more information. |
Fibronectin Interactions With Periodontal Ligament Cells @ University of California San Francisco
One of the major goals of periodontics has been the regeneration of a new connective tissue attachment following disease. The interactions of the periodontal ligament (PDL) fibroblasts with their extracellular matrix (ECM) and the changes associated with progression of periodontal disease and wound healing are complex processes which are likely to be particularly critical in understanding periodontal regeneration. The major goals of this study are to obtain a greater understanding of the role of fibronectin (FN) in the behavior of normal and diseased PDL fibroblasts and its role in tissue remodeling. We have chosen FN because of (1) its known importance in establishment of a stable pericellular matrix in many connective tissue cell types, (2) its known importance to the process of wound healing in other tissues such as skin, (3) its ability to promote migration of connective tissue cells, and (4) the possibility that degeneration of FN initiated by wounding or by the onset of periodontal disease, is likely to play a role in perpetuating the cycle of periodontal tissue destruction. We propose to characterize the production and stability of endogenous FN synthesized by normal PDL fibroblasts and to determine how PDL fibroblasts interact with exogenously added FN, FN isoforms, and different functional recombinant FN fragments. We will also test the hypotheses that the stability of FN produced by fibroblasts from diseased PDL is altered, and that the responses of diseased PDL fibroblasts to exogenous FNs differ from those of fibroblasts from normal PDL. Specifically, we will culture human PDL fibroblasts from normal and diseased tissues and analyze their endogenous FN at the protein and RNA level, using immunofluorescence and northern blotting techniques. The stability of endogenous FN produced by these cells will be analyzed by zymographic detection on substrate gels. To further characterize the interaction of FN with PDL fibroblasts, we will survey the FN receptors expressed on the surface on the PDL cells, using immunohistochemical and immunoprecipitation techniques. To determine how PDL fibroblasts interact with various forms of exogenously added FNs, we will use specific attachment and migration assays. Finally, to test our hypothesis that PDL fibroblasts from healthy and diseased tissue interact differently with FN, we will compare these cells by examining levels of expression of FN protein and mRNA, stability of FN protein and histology of the cells. From this study, we will gain a better understanding of the unique interactions that take place in the periodontium between PDL fibroblasts and the ECM, and of the effects of FN on the migration of PDL fibroblasts and their ability to synthesize a stable matrix. These studies relate directly to periodontal regeneration therapy and to wound healing in general.
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0.945 |
2001 — 2004 |
Kapila, Yvonne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apoptosis Regulated by Fibronectin and Its Receptors @ University of California San Francisco
The goal of these studies is to understand the mechanism by which proteolytic fragments of fibronectin (FN) mediate apoptosis in periodontal ligament (PDL) cells. Although the etiology of periodontal disease has been attributed to bacterial pathogens, several components in the pathogenesis of this disease remain poorly understood. One such area is the role of matrix fragments generated by bacterial proteinases and by inflammatory host-derive enzymes in the progression of periodontal disease. Proteases expressed by several putative periodontal pathogens readily cleave FN into multiple fragments, which are found in vivo and in association with periodontally disease sites. One such fragment is a 40 kDa chymotryptic fragment which contains the heparin-binding domain and part of the alternatively spliced V region of FN and can be generated by the chymotrypsin-like enzyme produced b Prevotella intermedia. Both this 40 kDa fragment and a longer recombinant (V+H-) fragment that also has the heparin-binding domain and the alternatively spliced V region of FN induce apoptosis in PDL cells. In addition, fragments of FN alter cell motility and enhance proteinase expression in PDL cells. These findings lead to the hypothesis that proteolytic fragments of FN generated by bacterial and/or host inflammatory cell proteinases affect several PDL cell functions including, survival, thereby exacerbating the degradation of periodontal tissues and contributing to disease progression. The specific aims are to: (1) Characterize the matrix parameters by which the 40 kDa FN fragment induces apoptosis in PDL cells; (2) Identify the cell surface receptors for the 40 kDa FN fragment, and for the 40 kDa-containing recombinant FN fragments that are involved in regulating apoptosis in PDL cells; and (3) characterize the signaling pathways by which the 40 kDa FN fragment and other fragments containing the 40 kDa region regulate apoptosis of PDL cells. These findings will contribute to our understanding of the pathogenesis of periodontal disease and to our basic understanding of the regulation of apoptosis by the extracellular matrix.
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2001 — 2005 |
Kapila, Yvonne L |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Pilot--Invasion Regulated by Fibronectin and Receptors @ University of California San Francisco
neoplasm /cancer genetics; neoplasm /cancer invasiveness; p53 gene /protein; tissue /cell culture
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0.945 |
2004 — 2009 |
Kapila, Yvonne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Fibronectin Regulation of Carcinoma Apoptosis @ University of Michigan At Ann Arbor
DESCRIPTION: Fibronectin (FN) and its receptors are important regulatory components in tumor cell survival. We have determined that the carboxyl-terminal heparin-binding domain and alternatively spliced V region of FN are important to this process, since a FN miniprotein (V+H-) containing a mutated heparin-binding domain and the V region of FN induces apoptosis of squamous cell carcinoma (SCC) cells. In contrast, the counterpart wildtype protein (V+H+) or other FN miniproteins not containing the V region promote survival of these cells. Furthermore, in SCC cells, the rate of V+H--mediated apoptosis is delayed compared to normal primary keratinocytes. These data suggest that tumorigenicity has enabled the SCC cells to delay their onset of apoptosis in response to an altered matrix, and that the V region and heparin-binding domain of FN regulate survival of these cells. Our preliminary data suggest that the mechanism by which the V+H- protein induces delayed SCC cell apoptosis may involve chondroitin sulfate proteoglycan and integrin receptors, and p53 and c-myc mediated signals. In primary fibroblasts, this apoptotic mechanism is mediated by a chondroitin sulfate proteoglycan, the alpha4 integrin, and by an intriguing new pathway that requires downregulation of p53 and c-myc. In addition, since p53 relocalizes from the nucleus to the cell membrane in this mechanism, and since the integrin-associated signaling molecule focal adhesion kinase (pp125FAK) and c-Jun N-terminal kinase (JNK) are depressed in this pathway, this suggests that p53 may communicate with integrin/FAK generated signals. We posit that SCC cells resist apoptosis in response to an altered FN matrix (V+H-) via cell surface proteoglycan and integrin receptors. This initiates a signal transduction pathway that leads to downregulation of FAK, p53 and c-myc. Secondarily, because of its important role in regulating SCC cell invasion, migration, and apoptosis, we posit that the V region of FN may be important to SCC cell pathogenesis. We will test these hypotheses in the following Specific Aims: (1) Identify the SCC cell-surface receptors involved in delaying apoptosis induced by the V+H- FN protein in these cells and compare these receptors to those present in primary keratinocytes. (2) Examine the SCC cell signaling response involved in delaying apoptosis induced by the V+H- FN protein. (3) Examine the expression of the alternatively spliced V region of FN in low grade and high-grade oral dysplasia and oral cancer. These studies will help explain some of the cell-matrix interactions and signaling mechanisms that regulate tumor cell biology, and may provide insights into the pathogenesis of oral SCC.
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1 |
2006 — 2010 |
Kapila, Yvonne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apoptosis Regulated by Fibronectin Signaling Pathways
Altered fibronectin matrices, as those elaborated during periodontal disease or inflammation compromise periodontal ligament (PDL)cell function. During the course of inflammation, bacterial and host-derived proteases cleave the extracellular matrix (ECM) and release fragments of the ECM, including fibronectin fragments, into the inflammatory milieu. We have shown that these fragments negatively influence PDL cell function by limiting their proliferative capacity and chemotaxis, and by inducing programmed cell death or apoptosis in these cells. The signaling mechanism by which this apoptosis is triggered is novel and requires the transcriptional downregulation of p53. Upstream of p53,decreases in focal adhesion kinase phosphorylation and increases in c-Jun N-terminal kinase (JNK)1 phosphorylation regulate this pathway. Furthermore, JNK1 and JNK2 oppositely regulate p53 levels in this process, however, the mechanism by which this occurs is not known. Yet, understanding this mechanism is critical to understanding how the stress-related condition of an altered fibronectin matrix environment regulates p53 and how p53 is regulated in general. Reports have shown that JNK phoshorylation of p53 can stabilize p53 and modulate its transcriptional activity. Conversely, JNK targets p53 for ubiquitination and proteasomal degradation in an Mdm-2 independent manner in nonstressed conditions. We hypothesize that JNK1 and JNK2 oppositely regulate p53 and apoptosis under altered fibronectin matrix conditions by modulating p53 at a transcriptional level and by ubiquitination and proteasomal degradation, independent of Mdm-2. Thus, the mechanism by which JNK1 and JNK2 oppositely regulate p53 and apoptosis under altered fibronectin matrix conditions will be examined in this study. This study will expand our understanding of the intricate mechanisms that tightly regulate p53,a key regulator of apoptosis, and of the specific modulation of p53 and apoptosis by stress-activated kinases,JNK1 and JNK2 under altered fibronectin matrix conditions which relate to periodontitis.
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2007 |
Kapila, Yvonne L |
M01Activity Code Description: An award made to an institution solely for the support of a General Clinical Research Center where scientists conduct studies on a wide range of human diseases using the full spectrum of the biomedical sciences. Costs underwritten by these grants include those for renovation, for operational expenses such as staff salaries, equipment, and supplies, and for hospitalization. A General Clinical Research Center is a discrete unit of research beds separated from the general care wards. |
Apoptotic Biomarkers of Periodontal Disease @ University of Michigan At Ann Arbor |
1 |
2010 |
Kapila, Yvonne L |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Fibronectin Regulation of Anoikis
DESCRIPTION (provided by applicant): Anoikis-apoptotic cell death triggered by loss of extracellular matrix (ECM) contacts is disregulated in many chronic debilitating and fatal diseases. In the autoimmune mucocutaneous diseases of pemphigus and pemphigoid excessive anoikis in the form of acantholysis or cell death by loss of cell-cell and cell-matrix contacts is the hallmark of these oral and skin lesions. In contrast, anoikis resistance contributes to cancer development and progression, and in oral squamous cell carcinoma (OSCC) anoikis resistance induces more aggressive tumors. Thus, regulating crosstalk between anoikis and survival signaling pathways is crucial to regulating tissue processes and mitigating diseases like autoimmune diseases and cancer. We recently reported that anoikis activates a CD95/Fas-mediated signaling pathway regulated by receptor-interacting protein (RIP), a kinase that shuttles between Fas-mediated cell death and integrin/FAK-mediated survival pathways in a variety of cell systems. Low RIP expression in OSCC compared to high expression levels in normal tissues underscored that RIP is at the crossroads of life and death pathways. However, it is not known how the ECM specifically interacts with the Fas receptor to mediate this mechanism and crosstalk, nor is it known how this is specifically regulated in vivo. Our preliminary data suggest that specific domains within the ECM protein fibronectin interact with the Fas receptor as the basis for this anoikis regulation. In addition, a Fas receptor mutation, which results in an inability of Fas to bind it's ligand, renders cells anoikis resistant in our system, further supporting a connection between the Fas receptor and specific domains of fibronectin. The fact that this Fas receptor mutation manifests as an autoimmune syndrome in humans (Canale-Smith syndrome) reaffirms the importance of apoptotic disregulation in autoimmune diseases. Since, there have been no reports of death receptors, like Fas interacting with ECM proteins, this constitutes a completely new area of investigation. On the survival side of the equation, we further found that sirtuins, a novel family of NAD- dependent deacetylases recently discovered to be involved in prolonging longevity and in cancer progression and metastasis, confer a survival advantage to OSCC cells. Thus, sirtuins may be important in promoting anoikis resistance in cells. Our global hypothesis is that disregulation of anoikis mediated by Fas- fibronectin interactions is central to many disease processes. Our specific hypothesis is that there are direct Fas-fibronectin interactions that mediate crosstalk with integrins to regulate anoikis via downstream signaling to RIP. Furthermore, anoikis resistant cells bypass this signaling pathway and favor sirtuin signaling to support their survival and a more aggressive phenotype in vivo. Knowledge gained from these investigations will help decipher novel interactions between matrix proteins and cell death receptors, and expand our understanding of life and death signaling networks. These data may help identify critical therapeutic targets for diseases like autoimmune diseases and oral cancer.
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1 |
2013 — 2014 |
Kamarajan, Pachiyappan Kapila, Yvonne L Rajendiran, Thekkelnaycke |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Biomarkers of Aggressive Oral Cancer
DESCRIPTION (provided by applicant): Anoikis-apoptotic cell death triggered by loss of extracellular matrix (ECM) contacts-is dysregulated in many diseases. Anoikis resistance contributes to the development and progression of cancer, thus marking aggressive tumorigenic transitions. In oral/head and neck squamous cell carcinoma (HNSCC), anoikis resistance induces more aggressive tumors. Given that oral SCC, the most common malignant oral neoplasm, accounts for 90% of all oral malignancies and has a poor 5-year survival rate that has not changed in decades (http://seer/cancer.gov), this underscores the need to identify novel therapeutic targets for HNSCC. Identifying markers of aggressive tumorigenesis and thus anoikis resistance could yield potentially novel therapeutic targets for HNSCC. Mass spectrometry-based metabolomics offers an innovative non-invasive platform for the development of marker panels that are characteristic of disease phenotypes or cellular processes that are readily measured in biofluids/tissues. We recently performed NMR based metabolomic analysis of primary and metastatic HNSCC human specimens and found elevated levels of several metabolites (Somashekar et al., 2011). These metabolites were associated with highly active glycolysis and glutaminolysis, increased amino acid influx into the Krebs cycle, and altered energy metabolism, membrane choline phospholipid metabolism, and oxidative/osmotic defense mechanisms. Using NMR spectroscopy, several groups also generated a list of similar metabolites differentially expressed across tissue and blood samples from head and neck cancer patients, (Mukheriji et al., 1997; Tiziani et al., 2009; El-Sayed et al., 2002). In complementary preliminary data where we now used mass spectrometry analyses of primary HNSCC, we identified several metabolites that were differentially and significantly elevated in HNSCC compared to normal tissues. Importantly, these metabolites were also elevated in metastatic HNSCC and in mouse tumors derived from anoikis-resistant HNSCC cells. These metabolites included kynurenine, serine, glutathione, and glutamate/glutamic acid. Since these metabolites have also been implicated in cancer metabolic pathways they constitute potential candidates for a marker panel of aggressive HNSCC. Furthermore, compared to normal oral keratinocytes, HNSCC cells also show elevated levels of glutamic acid. Elevated glutamic acid levels were suppressed in HNSCC cells by chemically inhibiting the enzyme that catalyzes the formation of glutamic acid levels, glutaminase. Inhibiting glutamic acid levels in these cells led to suppression of anoikis resistance/an aggressive phenotype in vitro. In addition, significantly elevated expression levels of glutaminase were highest in the metastatic tissues compared to primary HNSCC and normal tissues. These findings suggest that these metabolites, including glutamic acid may be markers for the transition to a more aggressive phenotype in cancer, including the acquisition of anoikis resistance. Our data support the concept that identification of a marker panel of metabolites present in aggressive HNSCC, a phenotype that emanates in part from anoikis resistance, may be useful as a new diagnostic tool and for identification of novel therapeutic targets for aggressive HNSCC. Thus, we hypothesize that aggressive states in HNSCC and anoikis-resistant HNSCC cells/tissues exhibit a metabolomic profile defined by increased levels of key metabolites that may include kynurenine, glutamic acid, serine, and glutathione. These metabolites mark aggressive tumorigenesis, the transition to anoikis resistance, and may promote anoikis resistance and an aggressive phenotype in vivo. These studies will help establish a signature metabolome or a panel of key metabolites for aggressive HNSCC and anoikis resistance plus validate the functional contribution of these metabolites in this process.
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1 |
2015 — 2019 |
Fenno, J Christopher Kapila, Yvonne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Oral Treponema Surface Proteins: Host Cell Interactions
? DESCRIPTION (provided by applicant): The goal of this research is to characterize the role of Treponema denticola (Td) surface proteins in interaction of this human oral spirochete with host tissue, thereby gaining insight into mechanisms by which these organisms contribute to initiation and progression of periodontal disease. We focus on analysis of Td protein complexes that directly affect cells isolated from tissue comprising the periodontal ligament (PDL) that comprises the junction between the tooth and the alveolar bone of the tooth socket: specifically the PrtP lipoprotein protease complex (dentilisin) and the oligomeric Msp protein. Our overall hypothesis is that dentilisin and Msp are major contributors to Td cytopathic behavior in periodontal disease. To characterize their specific roles in microbe-host interactions, our approach is to utilize purified native and recombinant proteins as well as isogenic Td strains carrying defined mutations in individual components of these outer membrane complexes. We will first extend our ongoing studies characterizing dentilisin assembly in the Td outer membrane. Then, to further studies of host cell responses to Td challenge, we will (A) determine the mechanism of dentilisin-induced activation of pro-MMP-2 and (B) characterize MMP-2-dependent fibronectin fragmentation resulting from Td challenge of PDL cells. This project will test the hypothesis that dentilisin is an effector protein whose activity results in magnified downstream signaling effects that are likely to be significant in vivo. Our research team is uniquely positioned to conduct these studies, with combined expertise in spirochete molecular biology, extracellular matrix biology and cytopathology of inflammatory diseases. Completion of this project will contribute to both basic knowledge of spirochete molecular biology and to understanding of microbe-host interactions in chronic infections such as periodontal diseases.
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1 |
2019 |
Fenno, J Christopher Kapila, Yvonne L |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Oral Treponema Surface Proteins: Host Cell Interactions - Diversity Supplement @ University of Michigan At Ann Arbor
ABSTRACT The goal of this research is to characterize the role of Treponema denticola (Td) surface proteins in interaction of this human oral spirochete with host tissue, thereby gaining insight into mechanisms by which these organisms contribute to initiation and progression of periodontal disease. We focus on analysis of Td protein complexes that directly affect cells isolated from tissue comprising the periodontal ligament (PDL) that comprises the junction between the tooth and the alveolar bone of the tooth socket: specifically the PrtP lipoprotein protease complex (dentilisin) and the oligomeric Msp protein. Our overall hypothesis is that dentilisin and Msp are major contributors to Td cytopathic behavior in periodontal disease. To characterize their specific roles in microbe-host interactions, our approach is to utilize purified native and recombinant proteins as well as isogenic Td strains carrying defined mutations in individual components of these outer membrane complexes. We will first extend our ongoing studies characterizing dentilisin assembly in the Td outer membrane. Then, to further studies of host cell responses to Td challenge, we will (A) determine the mechanism of dentilisin-induced activation of pro-MMP-2 and (B) characterize MMP-2-dependent fibronectin fragmentation resulting from Td challenge of PDL cells. This project will test the hypothesis that dentilisin is an effector protein whose activity results in magnified ?downstream? signaling effects that are likely to be significant in vivo. Our research team is uniquely positioned to conduct these studies, with combined expertise in spirochete molecular biology, extracellular matrix biology and cytopathology of inflammatory diseases. Completion of this project will contribute to both basic knowledge of spirochete molecular biology and to understanding of microbe-host interactions in chronic infections such as periodontal diseases.
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