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


    ARTICLE
TOP
ARTICLE
REFERENCES

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-beta (TGF-beta ) 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-beta -mediated increases in tropoelastin are achieved without affecting the rate of gene transcription. Instead, TGF-beta 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-zeta , 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.


    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).


    REFERENCES
TOP
ARTICLE
REFERENCES

1.   Bruce, MC, and Honaker CE. Transcriptional regulation of tropoelastin expression in rat lung fibroblasts: changes with age and hyperoxia. Am J Physiol Lung Cell Mol Physiol 274: L940-L950, 1998[Abstract/Free Full Text].

2.   Carreras, I, Rich CB, Jaworski JA, DiCamillo SJ, Panchenko MP, Goldstein R, and Foster JA. Functional components of basic fibroblast growth factor signaling that inhibit lung elastin gene expression. Am J Physiol Lung Cell Mol Physiol 281: L766-L775, 2001[Abstract/Free Full Text].

3.   Conn, KJ, Rich CB, Jensen DE, Fontanilla MR, Bashir MM, Rosenbloom J, and Foster JA. Insulin-like growth factor-1 regulates transcription of the elastin gene through a putative retinoblastoma control element. J Biol Chem 271: 28853-28860, 1996[Abstract/Free Full Text].

4.   Das, M, Dempsey EC, Bouchey D, Reyland ME, and Stenmark KR. Chronic hypoxia induces exaggerated growth responses in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase c isozymes. Am J Respir Cell Mol Biol 22: 15-25, 2000[Abstract/Free Full Text].

5.   Dietz, HC, and Mecham RP. Mouse models of genetic diseases resulting from mutations in elastic fiber proteins. Matrix Biol 19: 481-488, 2000[ISI][Medline].

6.   Foster, JA, Rich CB, and Miller MF. Pulmonary fibroblasts: an in vitro model of emphysema. Regulation of elastin gene expression. J Biol Chem 265: 15544-15549, 1990[Abstract/Free Full Text].

7.   Ghayor, C, Herrouin JF, Chadjichristos C, Ala-Kokko L, Takigawa M, Pujol JP, and Galera P. Regulation of human COL2A1 gene expression in chondrocytes. J Biol Chem 275: 27421-27438, 2000[Abstract/Free Full Text].

8.   Ichii, T, Koyama H, Tanaka S, Kim S, Shioi A, Okuno Y, Raines EW, Iwao H, Otani S, and Nishizawa Y. Fibrillar collagen specifically regulates human vascular smooth muscle cell genes involved in cellular responses and the pericellular matrix environment. Circ Res 88: 460-467, 2001[Abstract/Free Full Text].

9.   Kucich, U, Rosenbloom JC, Abrams WR, Bashir MM, and Rosenbloom J. Stabilization of elastin mRNA by TGF-beta: initial characterization of signaling pathways. Am J Respir Cell Mol Biol 17: 10-16, 1997[Abstract/Free Full Text].

10.   Majello, B, De Luca P, and Lania L. Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains. J Biol Chem 272: 4021-4026, 1997[Abstract/Free Full Text].

11.   McGowan, SE. Influences of endogenous and exogenous TGF-beta on elastin in rat lung fibroblasts and aortic smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 263: L257-L263, 1992[Abstract/Free Full Text].

12.   McGowan, SE, Jackson SK, Olson PJ, Parekh T, and Gold LI. Exogenous and endogenous transforming growth factors-beta influence elastin gene expression in cultured lung fibroblasts. Am J Respir Cell Mol Biol 17: 25-35, 1997[Abstract/Free Full Text].

13.   Parks, WC. Posttranscriptional regulation of lung elastin production. Am J Respir Cell Mol Biol 17: 1-2, 1997[Free Full Text].

14.   Parks, WC, Kolodziej ME, and Pierce RA. Phorbol ester-mediated downregulation of tropoelastin expression is controlled by a posttranscriptional mechanism. Biochemistry 31: 6639-6645, 1992[ISI][Medline].

15.   Pickering, JG. Regulation of vascular cell behavior by collagen. Form is function. Circ Res 88: 458-459, 2001[Free Full Text].

16.   Rich, C, Ewton DZ, Martin BM, Florini JR, Bashir M, Rosenbloom J, and Foster JA. IGF-I regulation of elastogenesis: comparison of aortic and lung cells. Am J Physiol Lung Cell Mol Physiol 263: L276-L282, 1992[Abstract/Free Full Text].

17.   Rich, CB, Fontanilla MR, Nugent M, and Foster JA. Basic fibroblast growth factor decreases elastin gene transcription through an AP1/cAMP-response element hybrid site in the distal promoter. J Biol Chem 274: 33433-33439, 1999[Abstract/Free Full Text].

18.   Richardson, TP, Trinkaus-Randall V, and Nugent MA. Regulation of basic fibroblast growth factor binding and activity by cell density and heparan sulfate. J Biol Chem 274: 13534-13540, 1999[Abstract/Free Full Text].

19.   Swee, MH, Parks WC, and Pierce RA. Developmental regulation of elastin production: expression of tropoelastin pre-mRNA persists after down-regulation of steady-state mRNA levels. J Biol Chem 270: 14899-14906, 1995[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 281(4):L762-L765
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society




This Article
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Stenmark, K. R.
Articles citing this Article
PubMed
PubMed Citation
Articles by Stenmark, K. R.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online