Areas of Interest
Research in the Shapiro laboratory concentrates on
understanding the regulation and function of cell surface
peptidases in the angiogenic endothelium of tumors, vessels at
sites of inflammation 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
We will extend the laboratory’s observations on the roles of
cell surface peptidases in various pathologic states. We have
conditional CD13 knockout animals and complete PSMA knockouts,
so both in vitro and in vivo studies are feasible. 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.
Lab Rotation Projects
CD13
- Investigation of signal transduction pathways induced by
CD13 ligation in endothelial cells that lead to increased
cell adhesion and their roles in inflammatory leukocyte
trafficking.
- Structure/function analysis of CD13’s signaling and
adhesion functions using chimeric mouse/human molecules.
- Characterization of the role of CD13 in inflammation in
response to bacterial infection.
- Characterization of CD13’s participation in inflammatory
disease models.
Investigation of CD13’s contribution to leukocyte
trafficking in inflammation.
- Molecular dissection of the reorganization of the
monocyte cytoskeleton following CD13 ligation.
- Assess the role of CD13 as an adhesion molecule of
endothelial junctions. CD13 relocates to the cell-cell
junctions as cells become confluent, suggesting it
participates in junction formation and endothelial
permeability.
- Characterization of the role of upregulated CD13
expression following myocardial ischemia in mouse models of
myocardial infarction (in collaboration with Bruce Liang,
Calhoun Center for Cardiology).
- Investigation into serum CD13 as a biomarker in
inflammatory and cardiovascular disease.
PSMA
- Structure/function analysis of PSMA’s regulation of beta
1 integrin signaling.
- Assessing PSMA as an endothelial adhesion molecule.
- Assessing endothelial PSMA in the angiogenesis during
wound healing.
- Investigating the role of PSMA in prostate cancer
metastasis. PSMA regulates prostate cancer cell invasion
suggesting it may regulate escape from the primary tumor and
access to metastatic sites.
- Investigation into how PSMA expression on tumor blood
vessels affects endothelial/basal lamina interactions and
vessel permeability.
Selected Publications
Mina-Osorio P, Winnicka B, O'Conor C, Grant CL, Vogel LK,
Rodriguez-Pinto D, Holmes KV, Ortega E, Shapiro LH. CD13 is a
novel mediator of monocytic/endothelial cell adhesion. J Leukoc
Biol. 2008 May 21. [Epub ahead of print]
Mahoney KM, Petrovic N, Schacke W, Shapiro LH. CD13/APN
transcription is regulated by the proto-oncogene c-Maf via an
atypical response element. Gene. 2007 403:178-87.
Petrovic N, Schacke W, Gahagan JR, O'Conor CA, Winnicka B,
Conway RE, Mina-Osorio P, Shapiro LH. CD13/APN regulates
endothelial invasion and filopodia formation. Blood. 2007
110:142-50.
Conway RE, Petrovic N, Li Z, Heston W, Wu D, Shapiro LH.
Prostate-specific membrane antigen regulates angiogenesis by
modulating integrin signal transduction. Mol Cell Biol. 2006
26(14):5310-24.
Fontijn D, Duyndam MC, van Berkel MP, Yuana Y, Shapiro LH,
Pinedo HM, Broxterman HJ, Boven E. CD13/Aminopeptidase N
overexpression by basic fibroblast growth factor mediates
enhanced invasiveness of 1F6 human melanoma cells. Br J Cancer.
2006 94:1627-36.
Mina-Osorio P, Shapiro LH, Ortega E. CD13 in cell adhesion:
aminopeptidase N (CD13) mediates homotypic aggregation of
monocytic cells. J Leukoc Biol. 2006 79:719-30.
Wentworth DE, Tresnan DB, Turner BC, Lerman IR, Bullis B,
Hemmila EM, Levis R, Shapiro LH, Holmes KV. Cells of human
aminopeptidase N (CD13) transgenic mice are infected by human
coronavirus-229E in vitro, but not in vivo. Virology. 2005
335:185-97.
Revised
August, 2008. |
