Cell-Surface Interactions in Pathogenesis

National Institute Of Dental & Craniofacial Research

Investigators

Linked publications, trials & patents

Abstract

Our goal in this project is to characterize new roles for cellular and cell-extracellular interactions in disease pathogenesis with a goal of identifying novel molecular regulators and mediators. Because we have extensive expertise in real-time imaging of cell behavior in vitro and in organ explants, we have an ongoing collaboration with the laboratory of Dr. Ashok Kulkarni in the NIDCR. We have refined methodologies to directly visualize craniofacial pain-associated signaling by live-animal real-time intravital microscopy using the genetically encoded sensor GCaMP6. We confirmed the role of Cdk5 in facial pain signaling and that a peptide inhibitor of Cdk5 was able to suppress elevated calcium signaling in response to both noxious stimuli and inflammation-induced allodynia by inhibiting the number and type of trigeminal peripheral sensory neurons that were activated. We are establishing a model for chronic neuropathic pain to compare mechanisms of trigeminal ganglion signaling in chronic rather than acute pain. Another ongoing area of focus is cancer invasion. We have been using both single cell migration and 3D spheroid systems to elucidate mechanisms of invasion across the basement membrane and within a 3D extracellular matrix, often a collagen hydrogel. We are studying both invasion across a basement membrane and individual cell migration under different physical conditions. Osteoarthritis is a highly prevalent joint disorder in the United States, with approximately 10% of men and 13% of women being afflicted by this condition. There are no approved treatments available to prevent or slow progression of the disease, with the only options being pain management and surgical intervention in severe cases. A prominent feature of osteoarthritis pathology is the breakdown of cartilage, which leads to the generation of matrix fragments through the activation of pathological integrin signaling. This integrin signaling plays a crucial role in mediating the production of proteases responsible for breaking down extracellular matrix components into fragments. However, the specific intracellular mechanisms through which integrin receptors transmit signals to induce the generation of MMP-13, a key protease involved in osteoarthritis, has been unclear. An ongoing collaboration with the laboratory of Prof. Richard Loeser, Director of the University of North Carolina Thurston Arthritis Research Center, is using primary human articular chondrocytes and human tissue from normal and osteoarthritic joints to provide insight into this disease. We found that primary human articular chondrocytes exhibit nonphagocytic internalization of a fibronectin matrix protein fragment in association with the alpha 5-beta 1 integrin. This internalization event triggers the formation of signaling redoxosomes, which are cell organelles actively involved in redox signaling. These redoxosomes generate localized intracellular gradients of hydrogen peroxide, leading to selective oxidation of specific protein targets within distinct microenvironments. Subsequently, this localized oxidative environment induces the production of proteases. The pathway therefore consists of fibronectin fragment binding to alpha 5-beta 1 integrin, endocytosis, H2O2 signaling from redoxosomes, induction of MMP-13, and further matrix degradation as the pathological cycle continues. Further direct analysis of human osteoarthritic cartilage confirmed the presence of redoxosomes during osteoarthritis pathogenesis. This knowledge identifies potential avenues for the development of targeted therapeutic strategies aimed at interrupting this self-amplifying cycle of matrix degradation to mitigate progression of this debilitating disease. We are continuing our research collaboration with Prof. Simon Tran in the Faculty of Dental Medicine and Oral Health at McGill University to provide approaches to prevent the loss of salivary gland function in patients receiving therapeutic radiation for head and neck cancer. After establishing that cellular extracts of human bone marrow show good efficacy against loss of salivary gland function in irradiated mice, the biologically active cell fraction was determined to be the mononuclear cell fraction. The granulocyte fraction was weak and induced an acute inflammatory response, and the red blood cell fraction was negative. We are continuing to identify active fractions to provide protection against salivary gland radiation damage and loss of function. Management of an active research laboratory entails a range of challenges in making ethical decisions that can be compounded by the various roles that a principal investigator must fulfill in terms of maintaining research productivity, mentoring trainees, obtaining ongoing funding support, organizing data for manuscript submission, successfully responding to requests for additional information from journal reviewers, and other roles. Some of these roles have the potential to come into conflict, which requires careful ethical management. In a collaboration with a team led by Dr. David Resnik at the NIEHS and NIH IRP Office of the Director, we outlined these potential conflicts and proposed ethical approaches to their management. From an alternative viewpoint, a trainee in our laboratory worked with collaborators to explore various perspectives of students and faculty for defining success in dental and healthcare education.