Research Interests
Research in the Shapiro laboratory concentrates on understanding the regulation and function of cell surface peptidases in the angiogenic endothelium of tumors and in cardiovascular disease. Numerous cell surface peptidases are strikingly upregulated on angiogenic endothelial cells leading to the hypothesis that these may functionally cooperate in enzymatic cascades to regulate angiogenesis and endothelial cell function. While the angiogenic significance of proteases that cleave large proteins (such as the matrix metalloproteases) is well documented, increasing evidence supports a role for peptidases (metAP2, CD13, APA, PSMA) as angiogenic regulators as well. The fact that these enzymes metabolize small peptide substrates suggests that small molecule regulators of angiogenesis exist which have yet to be identified and whose mechanisms are unknown. Indeed, we have shown that individually, two peptidases, CD13 and PSMA are potent regulators of angiogenesis and our investigation of their regulatory mechanisms and their possible interaction is the current focus of the laboratory.
We have shown that inhibition of CD13/APN blocks endothelial morphogenesis and invasion and we continue to examine the molecular mechanisms responsible for its angiogenic regulatory capabilities, particularly its role in invasion. Recent studies from our lab suggest that CD13 regulates signal transduction pathways leading to invasion by participating in plasma membrane organization via its interaction with membrane cholesterol. Further investigations have indicated that CD13 also functions as an adhesion molecule where it mediates inflammatory cell interactions as well as endothelial/extracellular matrix interactions in a signal transduction dependent manner which has strong implications for regulation of inflammatory leukocyte trafficking and angiogenic cell invasion. Furthermore, CD13 is expressed on adult pluripotent stem cells and may play a role in stem cell trafficking as well. Finally, high levels of CD13 are found in the serum of patients with certain types of cancers and inflammatory diseases and we are currently investigating the mechanisms regulating its release from the cell surface and CD13’s utility as a serum biomarker of chemoprevention in breast cancer and myocardial infarction. We have recently produced a conditional CD13 knockout mouse and are actively characterizing CD13’s contribution to various physiologic and pathologic processes by specifically inactivating the gene in specific tissues.
Our investigation into the function of a second cell surface peptidase, PSMA has shown that this peptidase also regulates cell signaling, albeit by an apparently different mechanism. Investigation of PSMA’s regulation of endothelial cell adhesion led to the very interesting discovery that PSMA is a component of a complex regulatory loop that controls integrin signaling and PAK1 activation. Invasion studies with PSMA-null cells showed that PSMA regulates cell invasion by controlling signaling from beta1 integrins to focal adhesion kinase (FAK) and PAK1. We showed that PSMA interacts with the actin-binding protein filamin A, and disruption of this interaction decreases the peptidase activity of PMSA and phosphorylation of PAK1 in cultured cells. The interaction of PMSA with the cytoskeleton via filamin A allows a feedback signal from integrin beta1 and PAK that holds PMSA activity in check. Inhibition of PAK by expression of a peptide corresponding to its autoinhibitory domain enhanced the association of PMSA with filamin A, thus increasing its peptidase activity. These studies suggest an extracellular matrix derived, small molecule PSMA substrate that superactivates beta1 integrins, thus regulating angiogenesis and cell invasion. The manuscript describing this work was recently featured as a ‘highlight of the recent literature’ by the editors of Science.
Investigation into the regulation and function of these molecules will increase our understanding of molecular mechanisms controlling blood vessel formation in a variety of diseases such as cancer, heart disease, inflammatory disorders, diabetic retinopathy, and arthritis.
Future Directions
Linda Shapiro plans to extend her laboratory’s observations on the roles of cell surface peptidases in angiogenesis and cell invasion. We have conditional CD13 knockout animals and complete PSMA knockouts, so both in vitro and in vivo studies are planned. For CD13, experiments will be directed toward its role as an adhesion molecule on endothelial cells and monocytes and its potential regulation of inflammation, inflammatory diseases and cancer. Specific areas of research will elucidate its interacting adhesion partners, its place in the established paradigm of leukocyte trafficking, characterization of the signal transduction cascades induced by CD13 and investigation of its internalization and re-expression on the membrane. In addition, studies will continue on the mechanism of CD13 shedding and the function of soluble CD13 in inflammation and stem cell trafficking. Studies regarding PSMA will focus on its role as an angiogenic regulator and its control of cell invasion via integrin signaling. Specifically we will concentrate on identifying its angiogenic/integrin activating peptide substrate, the concept of regulation of angiogenesis by small extracellular matrix derived-peptide fragments and the role of PSMA in prostate tumorigenesis and invasion.
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