EDITORIAL FOCUS
Cell-, age-, and phenotype-dependent differences in the
control of gene expression
Kurt R.
Stenmark
Department of Pediatric Critical Care and Developmental Lung
Biology Laboratory, University of Colorado Health Sciences Center,
Denver, Colorado 80262
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ARTICLE |
ELASTIN IS AN ESSENTIAL
EXTRACELLULAR component of the pulmonary airways and vasculature
that acts, in part, to provide the elasticity necessary to sustain
respiratory and vascular dynamic functions (5, 13). The
bulk of elastin production and deposition is limited to a narrow window
of development, thus providing a somewhat unique model for the study of
the molecular mechanisms controlling production of an age- and
tissue-specific matrix protein. Elastin production in the lung begins
around midgestation, peaks near birth and during early neonatal
periods, drops sharply thereafter, and is nearly repressed by maturity
(1). Once produced, mature elastin is extremely durable
and essentially does not turnover in healthy tissues, and thus its
limited pattern of production does not normally compromise tissue
integrity or organ function over the lifespan of the individual.
However, certain lung diseases such as pulmonary hypertension,
scleroderma, and fibrotic and granulomatous lung diseases are
associated with increased production and excessive accumulation of
elastin, whereas other conditions such as bronchopulmonary dysplasia,
emphysema, and aneurysm are characterized by destruction or a marked
deficiency of elastin and, in some cases, by an inability to restore
this lost matrix in an appropriate tissue-specific way. In these
situations, excesses and/or deficiencies of elastin can severely
compromise the physiological structure and function of the affected
tissue. Therefore, it is extremely important to understand the
mechanisms that operate to control elastin production, especially in
disease states, so that therapies can be designed to restore elastic function.
In an effort to better understand the local mechanisms controlling
aberrant elastic fiber formation in pulmonary emphysema, the Foster
group developed and utilized over the years a neonatal rat
pulmonary fibroblast culture system as an in vitro model to study the
regulation of elastin gene expression (3, 6, 17). They
have shown that basic fibroblast growth factor (bFGF) downregulates elastin gene transcription in this system via the binding of a Fra-1/c-Jun heterodimer to an activator protein-1/cAMP response element
in the distal region of the elastin promoter and have suggested that
bFGF release by proteases may have important biological significance in
situations of pulmonary injury and/or repair (17). In this
issue of the American Journal of Physiology-Lung Cellular and
Molecular Physiology, Carreras et al. (2) elucidate
the intracellular signaling pathways through which bFGF downregulates elastin transcription. Interestingly, the investigation demonstrates that elastin expression and bFGF signaling are remarkably different between confluent and subconfluent fibroblast cultures. This is important because the studies suggest that certain repair mechanisms (at least those involving elastin) may be utilized in a spatially specific manner in the setting of elastase-induced lung injury. The
demonstration of marked differences in the regulation of elastin through second messenger signaling pathways in neonatal subconfluent versus confluent cells also has potential implications for those studying the regulation of other genes and suggests that cell type,
developmental age, and phenotypic state of the cell (confluent or
subconfluent, quiescent or proliferating, differentiated or dedifferentiated) must be considered when designing studies aimed at
elucidating the mechanisms regulating cell-specific gene expression.
The regulation of tropoelastin synthesis in the interstitial lung
fibroblast has been of particular interest because this cell is the
primary source of interstitial elastin in the alveolar wall
(1). Initial studies evaluating regulation of elastin in
the whole lung or other tissues were important but did not specifically
or adequately address control of tropoelastin expression in the cell
type of interest (interstitial fibroblast) because the regulatory
control of tropoelastin appears to vary with cell type during
development. For instance, in the postnatal lung, tropoelastin mRNA
levels (by in situ hybridization) appear to peak by about day
4 in vascular smooth muscle cells (SMCs) but not until day
11 in interstitial fibroblasts (1). Rich et al. (16) demonstrated that in vitro exposure to insulin-like
growth factor I increased tropoelastin mRNA and protein in aortic SMCs but not in lung fibroblasts from 2- to 3-day-old pups. Transforming growth factor-
(TGF-
) was shown by McGowan (11) to
increase steady-state tropoelastin mRNA and soluble elastin levels in
lung fibroblasts but not in SMCs. These observations suggest that
potentially significant differences in the control of elastin gene
expression exist between cell type(s) in an organ during development
and suggest that mechanisms utilized to regulate specific gene
expression may be cell type-specific.
In essentially all tissues and species, production of elastin
correlates with the steady-state levels of its mRNA (13). Tropoelastin gene transcription and message expression were initially thought to be coordinately regulated. However, Parks' laboratory has
challenged this concept. They (19) found in cultured adult lung fibroblasts that tropoelastin gene transcription is not
downregulated, although steady-state tropoelastin mRNA levels are low,
and demonstrated that termination of tropoelastin expression in adult
lung fibroblasts is under posttranscriptional control. Other studies
(9, 12, 13) support the concept that in postnatal
lung fibroblasts, elastin production is indeed influenced by
tropoelastin mRNA stability. Indeed, TGF-
-mediated increases in
tropoelastin are achieved without affecting the rate of gene
transcription. Instead, TGF-
1 mediates a marked stabilization of
tropoelastin mRNA, leading to increased steady-state transcript levels
and enhanced protein production that in lung fibroblasts is mediated by
a protein kinase C (PKC)-dependent pathway (9, 12). These
results differ from those previously reported (14) in
cultured bovine elastic chondrocytes where phorbol esters (specific
activators of PKC) actually downregulated elastin expression by
destabilizing tropoelastin mRNA. Interestingly, the PKC isozymes
involved in the tropoelastin mRNA stabilization pathway in lung
fibroblasts were unresponsive to phorbol ester, thereby clearly
distinguishing the PKC pathways operating in lung fibroblast
stabilization pathways from those acting in the fetal chondrocyte
system. These findings are perhaps not surprising because the 12 known
members of the PKC family have been shown to have different activation
properties, cofactor requirements, tissue and cell distributions, and
even compartmentalization differences within the cell. Furthermore, Das
et al. (4) recently demonstrated that the PKC isozymes
used in mediating hypoxia-induced proliferative responses in pulmonary
artery adventitial fibroblasts vary depending on the developmental
stage of the cell. Thus it seems certain that PKC isozymes are utilized
in a cell-specific as well as a developmental-specific manner and can
thus mediate different responses to similar stimuli. Collectively,
these observations demonstrate the existence of cell-specific and even
developmental-specific differences in the elastogenic response to
specific stimuli and in the signaling pathways utilized to achieve the
response. The study by Carreras et al. (2) emphasizes that
an understanding of the mechanisms regulating a specific gene in a
disease process must be achieved by utilizing the most specific and
relevant model cell system available.
Many of the published studies regarding bFGF and its effects on cell
proliferation, growth and differentiation, and the signaling pathways
associated with these responses have been performed in transformed cell
lines. The experiments by Carreras et al. (2) clearly
point out the importance of evaluating signaling pathways under
conditions in which no cellular transformation has taken place and the
importance of a cellular model system where the cells are in a flexible
state of phenotypic expression and whose entry into the cell cycle is
dependent on numerous factors including cell-matrix communication and
cell-cell contact. A previous study (18) has clearly
demonstrated that cell density, whether achieved by plating conditions
or culturing to a specific cell density, provides the cell with a
mechanism by which bFGF binding and signaling may be modulated. The
duration of bFGF-dependent extracellular signal-regulated kinase
signaling as well as the endogenous levels of activator protein-1
family members differ significantly between subconfluent and confluent
primary fibroblast cell cultures, a situation that would probably not
exist in stably transformed cell lines. The differences in
bFGF-initiated signaling could be attributable to differences in
heparan sulfate proteoglycan (HSPG) expression by the cells because
HSPG expression modulates bFGF effects and depends on the cell
phenotype (18). HSPGs from cells at high density have
longer glycosaminoglycan chains than those from cells at lower cell
density. Furthermore, HSPGs derived from nondividing SMC cultures have
a 10-fold higher antiproliferative potency than HSPGs derived from
proliferating cells, observations all confirming a strong correlation
between cell phenotype, differential HSPG expression, and bFGF
responses (18). Thus the findings by Carreras et al.
(2) demonstrating that bFGF modulates specific cellular
events depending on the growth and density status of the cell provide a
rationale for the differential cellular responses that are observed in
different sites within an organ in development and in injury or repair situations.
The idea that the differentiation state of a cell may significantly
affect the response of specific genes to transcription factors
has also been demonstrated in other cell types. Chondrocytes, for
instance, when isolated from cartilage tissue and cultured as a
monolayer, lose their cartilage phenotype and transform into fibroblast-like cells on repeated passages (7). Ghayor et
al. (7) investigated the mechanisms that regulate
transcriptional activity of the cartilage-specific type 2 procollagen
gene (COL2A1) in adult chondrocytes because it is a good marker of the
differentiated cell state. Interestingly, they showed that
overexpression of the transcription factor C-Krox, shown to activate
the COL2A1 gene in differentiated chondrocytes, actually inhibited
transcription of the collagen gene in dedifferentiated cells via
changes in binding in the promoter region of COL2A1. It is also worth
noting that C-Krox homologs such as Kruppel and Yin-Yang (YYI) have
also been shown to either activate or inhibit gene transcription
depending on whether they form homo- or heterodimers and in the context of where the gene is found (10). These observations are
consistent with those of Carreras et al. (2), where the
bFGF response element does not exhibit any complex formation with
nuclear proteins isolated from subconfluent fibroblast even though the
levels of Fra-1 are very high, suggesting that the ability of Fra-1 to
bind this element may depend on the level of heterodimer partners
(i.e., c-Jun and its competitor c-Fos) or its phosphorylation state. Thus certain transcription factors (e.g., C-Krox, Sp3, and perhaps Fra-1) could be considered as bifunctional transcriptional factors whose effects depend on the differentiation state of the particular cell in which they are expressed. Therefore, cell density and the
differentiation state of the cell will influence not only the signaling
pathways initiated in response to a specific stimulus but also how
transcription factors bind to and regulate the gene of interest.
Alterations in extracellular matrix production and accumulation that
vary depending on the differentiation state of the cell may also play a
significant role in modulating the response of a cell to specific
stimuli. In many lung diseases including bronchopulmonary dysplasia,
emphysema, and pulmonary hypertension, the extracellular matrix can
become quickly remodeled through a combination of synthesis of new
extracellular matrix molecules and proteolytic degradation and editing
of existing matrix molecules. Recent studies (8, 15)
demonstrated that even the form of collagen in the extracellular matrix
may have a tremendous influence on gene expression and cell behavior.
Fibroblasts grown on polymerized collagen, in contrast to those grown
on monomeric forms of collagen, stop replicating, reduce their
expression of type I and III collagens, induce the expression of
collagenase I, activate PKC-
, and exhibit attenuated responses to
growth factors. Ichii et al. (8) recently demonstrated the
profound effect that polymerized versus monomeric collagen has on gene
expression of vascular SMCs. The authors established that fibronectin,
thrombospondin, and tenacin C, all factors traditionally associated
with a proliferative phenotype, were downregulated by polymerized
collagen, highlighting a phenomenon whereby the extracellular matrix
environment in vitro (and most likely in vivo) can regulate its own
constituents. They also confirm that significant differences exist in
the ability of cells to respond to certain stimuli depending on the
type of collagen (matrix) they are grown on. The findings are
consistent with a mechanism whereby the extracellular matrix can
suppresses cellular responses to other matrix molecules or perhaps
other signaling molecules residing in the matrix, i.e., bFGF. Thus, as
suggested by many, a dynamic reciprocity exists between the cell and
the substrate on which it rests that regulates both the constituents of
the extracellular matrix and the responsiveness of the cell to those constituents (15). It thus appears that in addition to
developmental age and differentiated state of the cell, careful account
of the matrix on which the cells are initially cultured and the
matrix that is produced under different conditions must be taken into account when evaluating the effects of specific stimuli on a specific gene.
In summary, the Foster group had provided important and exciting
information regarding the hypothesis that bFGF-mediated control of
elastin expression is different within cells that are either adjacent
to or remote from elastase activity in the injured lung. This is
probably because the phenotypic state of the cell plays a large part in
dictating the response that is observed in response to a specific
extracellular signal. Complete elucidation of the mechanisms that
regulate elastin synthesis as well as other gene products in lung
disease processes such as emphysema and bronchopulmonary dysplasia will
require model systems that take into account the fact that regulation
of specific cellular responses to various stimuli will vary depending
on cell type, developmental age of the cell, cell differentiation
state, cell density, and the presence of specific matrix constituents.
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FOOTNOTES |
Address for reprint requests and other correspondence: K. R. Stenmark, Dept. of Pediatric Critical Care and Developmental Lung
Biology Laboratory, Univ. of Colorado Health Sciences Center, Denver,
CO 80262 (E-mail: kurt.stenmark{at}uchsc.edu).
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