Our Science – Roberts Website
David D. Roberts, Ph.D.
Regulation of Cellular Function and Gene Expression by Extracellular Matrix Components in Tumor Cells and Opportunistic Pathogens
Cell-cell and cell-matrix interactions are important regulators of normal cell growth and differentiation. They also play essential roles in pathological conditions such as tumor metastasis and microbial infections. We are defining functions of extracellular matrix molecules, their receptors, and the signal transduction pathways that regulate their activities in specific diseases. These studies will identify new molecular targets for intervention and thereby provide a basis for designing novel therapeutic agents. Currently, our primary research projects are: 1) investigating the mechanism by which thrombospondins regulate tumor angiogenesis, 2) developing a systems biology approach for understanding angiogenesis inhibitors, 3) investigating the regulation of immune and inflammatory responses by thrombospondin-1 (TSP1), and 4) identifying genes required for disseminated infections by the pathogenic yeast Candida albicans. These projects are in somewhat divergent fields but share the common goal to define the mechanisms by which cells communicate with extracellular matrix. The cross fertilization between these projects has helped us to make significant contributions to each field.
Regulation of angiogenesis by thrombospondins. We identified additional β1 integrin receptors that mediate pro-angiogenic activities of TSP1 and TSP2. Recombinant constructs containing these sequences stimulate angiogenesis, but peptide ligands of these receptors are novel angiogenesis inhibitors. We propose that TSP1 is a context-dependent modulator of angiogenesis and tumor perfusion via its effects on endothelial and vascular smooth muscle cells. We identified signaling pathways that control vascular cell responses to TSP1 and continue to study how opposing signals from vascular cell TSP receptors are integrated to control angiogenesis.
An important advance in our understanding of the anti-angiogenic activity of TSP1 arose from discovering the central role of cGMP in this response. Nitric oxide (NO) stimulates angiogenesis via cGMP signaling, and TSP1 potently inhibits this pathway via two TSP receptors. Surprisingly, ligation of the well known inhibitory receptor CD36 is sufficient but not necessary for TSP1 to inhibit NO/cGMP signaling in endothelial cells. In contrast, CD47 is the essential TSP1 receptor. We discovered that antagonism of NO signaling via CD47 extends to vascular smooth muscle cells, expanding the role of TSP1 in angiogenesis to coordinating endothelial and perivascular responses. Using functional imaging, we found that endogenous TSP1 is a potent regulator of tissue perfusion through its antagonism of NO signaling in vascular smooth muscle.
Systems biology of angiogenesis inhibitors. We proposed that critical signaling nodes for angiogenic responses could be identified that would provide new targets for developing therapeutic inhibitors of angiogenesis. Using mRNA and proteomics profiling, we compared the responses of endothelial cells to 4 dissimilar angiogenesis inhibitors. These complementary techniques allowed us to identify transcriptional as well as translational and post-translational targets of the angiogenesis inhibitors. Through this approach we found that phosphorylation of the cytoskeletal regulators cofilin and hsp27 is coordinately induced by 4 angiogenesis inhibitors. Growth regulatory genes were identified as a second signaling node, which is regulated at the mRNA level.
Regulation of T cell function by TSP1 and TSP2. Although the tumor suppressive activity of TSP1 was initially ascribed to inhibition of angiogenesis, we have obtained evidence for significant direct effects of TSP1 on T cell responses that are involved in anti-tumor immunity. Interactions of T cells with TSP1 are mediated by CD47, an unidentified heparan sulfate proteoglycan, and α4β1 integrin. Based on cDNA microarray analyses, TSP1 globally suppresses TCR signaling. Two TSP1 receptors, CD47 and heparan sulfate proteoglycan, mediate this activity. α4β1 integrin plays dominant roles in T cell adhesion, chemotaxis, and regulation of matrix metalloproteinase gene expression by TSP1 and TSP2. Our future research will examine how signals from these three TSP receptors are integrated to regulate T cell function. We are defining signal transduction pathways for each TSP receptor and identifying genes that are targets of these signals. Based on our microarray analyses, TSP1 targets include several early response genes in TCR signaling.
Abnormal immune responses have been reported in TSP1 or TSP2 null mice. To understand the molecular bases for these phenotypes, we are assessing changes in lymphocyte differentiation or functional responses in naive or challenged TSP1 null and wild type mice. We have identified TSP1-dependent molecular alterations in splenic and thymic lymphocytes. To complement these specific marker studies, we are using microarrays and proteomics to identify molecular alterations in T cells and lymphoid organs of the TSP1 null mice.
Host colonization and vascular dissemination of Candida albicans. Because clinical isolates of Candida sp. are increasingly resistant to the available antifungal agents, new approaches are needed to prevent and treat these infections in cancer patients. We found that hemoglobin specifically induces expression of a C. albicans receptor for the host matrix protein fibronectin. This response is mediated by a hemoglobin receptor and is conserved among pathogenic species in the Candida genus. We identified a hemoglobin-induced cell wall protein that binds to fibronectin and cloned several novel genes that are rapidly induced in cells exposed to hemoglobin in vitro or by incubation in the vascular compartment of rabbits. One of these genes, HBR1, is essential for vegetative growth and is haploinsufficient for regulation of mating type locus genes, white-opaque switching, and some stress responses. A second hemoglobin-induced gene was identified as a heme oxygenase, which is essential for growth on iron-limiting medium and metabolizes exogenous heme to α-biliverdin. These changes in gene expression define a new differentiation pathway by which C. albicans adapts to the vascular compartment of its host. Understanding the signal transduction pathways that regulate this differentiation pathway could lead to new therapeutic targets to manage disseminated candidemia.
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This page was last updated on 2/22/2013.