Research summary:
My laboratory focuses on the molecular mechanisms that
govern apoptosis and cell cycle and their relationship to the pathogenesis
of inflammatory disease, specifically rheumatoid arthritis (RA). During RA,
the tissue surrounding the cartilage and bone, the synovial lining, which
is comprised of fibroblasts and macrophages, increases from 1-2 layers thick
to as much as 10 or more layers through enhanced proliferation, migration,
and/or decreased cell death. Thus, our goal is to understand the pathways
that regulate fibroblast growth and survival and monocyte/macrophage differentiation,
migration and survival and how these pathways are applicable to the initiation
and progression of RA.
Figure 1. (A) Autoradiograph of bone in an RA patient. Arrowheads
denote areas of bone destruction. (B) Schematic representation of the bone-cartilage
junction showing pannus formation (invading synovial lining). Picture is adopted
from Pope et al. Nature Reviews Immunology 2002, 2: 1-9. (C) Histologic examination
demonstrating induction of experimental adjuvant-induced arthritis in rats.
Ankle sections were stained with hematoxylin (blue) and eosin (pink). For
abbreviations; C=cartilage; SL=synovial lining; B=bone; P= pannus. Black arrows
denote sites of invading pannus. Picture was adopted from Perlman et al. Arthritis
& Rheumatism 2001, 44:2899-2908
Fibroblast Projects
We demonstrated that the anti-apoptotic protein Bcl-2, is highly expressed
in RA and correlates with disease activity. We developed a gene delivery technique
to suppress Bcl-2 expression by employing a replication defective adenovirus
expressing a ribozyme against Bcl-2 mRNA. Unlike Bcl-2 deficient cells, the
forced down regulation of Bcl-2 in fibroblasts induces apoptosis and early
entry into S-phase of the cell cycle. Thus, our future studies will entail
examining which factors in the mitochondrial apoptotic pathway are required
for forced Bcl-2 ablation induced cell death. We will also examine whether
aberrant cell cycle entry following Bcl-2 reduction is required or a byproduct
of apoptosis. Lastly, we will examine whether suppression of Bcl-2 is a viable
therapy for ameliorating experimental arthritis development.
A second project is to examine the role of cell cycle regulatory proteins
in RA fibroblasts. We demonstrated that p21 and Rb, two negative regulators
of cell cycle, inhibit the pro-inflammatory molecules IL-6 and MMP-1 in RA,
but not in normal joint fibroblasts. Thus, our future plan is to examine how
p21 and Rb regulate IL-6 and MMP in RA fibroblasts. We will also examine mice
deficient for cell cycle inhibitory proteins and determine whether the loss
of these molecules results in the spontaneous induction of arthritis or exacerbates
experimental arthritis development.
Macrophage Project
We demonstrated that the prototypic death receptor-ligand pathway, Fas-FasL
regulates peripheral blood derived monocyte survival in culture even in the
absence of growth factors. Furthermore, the direct Fas-FasL inhibitor Flip
is upregulated during monocyte activation and during monocyte-macrophage differentiation
thereby suppressing the Fas-FasL death signal. To this end, our goal is to
determine if deficiencies in Fas or FasL contribute to monocyte survival in
the bone marrow and circulation, and differentiation into a macrophage by
employing mice deficient for Fas (lpr) and FasL (gld). Additionally, we are
examining whether the loss of Fas can restore normal monocyte numbers in M-CSF
mutant mice (op/op), which display reduced number of monocytes in the bone
marrow and in the circulation and fewer resident macrophages, indicating that
M-CSF is required form monocyte survival. Since we also demonstrated that
Flip is upregulated during monocyte activation and during differentiation,
we will transfer fetal liver cells from Flip-deficient mice, which die in
utero (day 9) into lethally irradiated hosts to examine whether Flip is required
for monocyte activation, monocyte-macrophage differentiation and/or macrophage
survival. Lastly, we will determine whether constitutive endothelial FasL
expression reduces monocyte survival, blocks monocyte extravasation and suppresses
the development of experimental arthritis by breeding mice lacking FasL with
transgenic mice that have been created to constitutively express FasL under
the control of an endothelial cell specific promoter. Collectively, these
experiments employing knockout and transgenic mice will elucidate the factors
that regulate monocyte homeostasis under normal conditions and will be applicable
to the study of RA.