PERSPECTIVE
Smad about Elastin Regulation

Jeffrey M. Davidson

Department of Pathology, Vanderbilt University School of Medicine and Department of Veterans Affairs Medical Center, Nashville, Tennessee

Elastin is crucial to pulmonary function, yet it appears in the pulmonary vasculature and interstitium late in gestation in large mammals and postnatally in rodents (1). Deletion of the ELN gene in mice produces obstructive arterial disease (2, 3) and disrupts terminal airway branching as well as alveogenesis (4); moreover, several forms of elastic tissue disease collectively known as cutis laxa have mild to fatal pulmonary complications (5). Once elastin has been deposited together with other elements of the elastic fiber, elastin synthesis ceases and turnover is close to nil. However, a program of neosynthesis of elastin can be rapidly activated by pulmonary hypertension and lung injuries leading to fibrosis (6, 7). Among the most potent modulators of elastin production is the pleiotropic growth factor, transforming growth factor (TGF)-.

In this issue, Kucich and coworkers report several novel observations that broaden yet complicate the signal mechanisms by which TGF- exerts its effects on elastin mRNA stability (8). Signaling from the heteromeric, serine-threonine kinase TGF- receptors (TGF-Rs) at the cell surface causes inhibition of epithelial proliferation, epithelial-mesenchymal transformation, chemoattraction of inflammatory cells, immunosuppressive effects, and a particular group of responses that affect extracellular matrix metabolism and recognition. Much interest and excitement has been created by the identification of the Smad group of transducing factors (a condensation of the terms for two gene families involved in TGF- superfamily signaling: Sma from Caenorhabditis elegans and mothers against decapentaplegic in Drosophila). The Smad group of transducers, cotransducers, and counter-transducers can transmit signals from the TGF-Rs directly to nuclear transcription targets. The direct action of these factors on transcriptional machinery has offered relief from the complex networks involved in signaling from tyrosine kinase growth factor receptors such as those recognizing epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. It's not that simple. Kucich and colleagues, in a series of papers, have presented persuasive data showing that the ultimate effects of TGF- on elastin, fibronectin, and CTGF expression require other downstream signaling pathways (8). These data address the concept of TGF- signaling that are not intrinsic to the Smad pathway.

The end points in the current paper by Kucich and coworkers are the accumulation of elastin mRNA and secretion of tropoelastin by lung fibroblasts after TGF- treatment. Elastin transcription may be modestly affected by TGF- treatment in some cells, but a series of studies over the past 10 years have implicated mRNA stability as the primary mechanism that regulates elastin mRNA levels and elastin synthesis in connective tissue cells (14). TGF- can affect the mRNA stability in a number of other connective tissue genes, including COL1 (18), fibronectin (19), and RHAMM (20). Furthermore, the stability of nonmatrix transcripts such as ribonucleotide reductase R2 appears to be regulated by TGF- (21, 22). At least some of the effects of TGF- on matrix accumulation are not due to direct action of Smads on matrix genes. This point is amplified and clarified by the present study.

Elastin is one of a few proteins whose expression is extensively controlled at a post-transcriptional level (1). Parks and colleagues performed a number of studies on elastin regulation that emphasized the impact of mRNA degradation on regulation of elastin mRNA by vitamin D, steroids, and phorbol ester (24). Following on the observation that TGF-1 was a strong stimulus for elastin expression (23), Kahari and coworkers showed that this effect was largely due to changes in mRNA stability (15). In contrast, studies of transcriptional regulation of elastin synthesis have identified only a limited number of regulatory factors, some of them negative, and most having a narrow dynamic range (27). The elastin promoter does show modulation by IGF-1 (28), and a transgenic mouse strain using a 6-kB human elastin promoter shows reasonably tight temporal and tissue-specific regulation (31).

In the developing lung, elastin accumulation sharply rises during the phase of alveolarization, and accumulation is reflected in large increases in elastin mRNA abundance. Studies by Swee and coworkers suggest that elastin transcription is activated during this stage of differentiation, and elastin mRNA stability is high (32). At the cessation of lung development, elastin synthesis and elastin mRNA levels fall to nearly undetectable levels, consistent with the very low turnover rates of elastin during adult life. However, estimates of elastin transcription based on nuclear pre-mRNA suggest that transcription levelsat least in rat lung fibroblastsremain high throughout adult life, implying that mRNA decay is the predominant regulatory mechanism in the rat lung fibroblast. Thus, the role of TGF- and other modulators of RNA stability becomes an important aspect of pulmonary physiology.

TGF- is known to modulate mRNA stability in other systems. For example, the 3' UTR of ribonucleotide reductase R2 reportedly contains a GAGUUUGAG sequence that specifically interacts with a 75 kD complex to enhance mRNA stability (22, 33). A similar phenomenon has been described for the hyaluronan receptor, RHAMM (20). In distinction from this element, Zhang and colleagues described a sequence from the coding region of rat elastin mRNA that is transcribed from ELN exon 30 (34). Unlike the behavior of the 3'UTR element, TGF- appears to reduce the binding of a cytosolic, trans-acting protein whose interaction with mRNA destabilizes elastin transcripts. Deletion studies of a core oligonucleotide sequence show that mutations in this area will reduce protein binding, leading to increased mRNA stability. In the human, exon 30 is a much shorter sequence, and two cases of point mutations in the 5' portion of exon 30 lead to a remarkable loss of elastin mRNA stability associated with the phenotype of autosomal dominant cutis laxa (35). TGF- has a dramatic effect on elastin mRNA stability in skin fibroblasts from these two cutis laxa strains (35, 36). Elastin mRNA half-life shifts from a few minutes to many hours, the net result of which is a large increase in elastin mRNA concentration and elastin synthesis. It is conceivable that the observations in rat and human elastin transcripts are related, but there is no data regarding RNA-protein interactions of the human transcript. In the case of elastin mutations, cycloheximide prevented increased elastin mRNA degradation after TGF- withdrawal.

Kucich and coworkers show that there is a considerable lag (4-6 h) between the addition of TGF- and the response of increased mRNA levels, consistent with earlier findings at the level of elastin synthesis (8), and protein synthesis is required for at least 4 h. Some of the delay might be attributable to the machinery of mRNA degradation, but the data certainly suggest that a series of events transpire before mRNA accumulation begins. The Smad pathway is a key element, because transfection of the inhibitory Smad7 blocks TGF--stimulated elastin mRNA accumulation. However, the data imply that Smad signaling is necessary but not sufficient. The collective studies of this group show that critical signals include: geranylgeranylation of an unknown target (a small, non-Ras, GTPase) (16); acyl transferase (16); and the action of kinases SAPK and PKC- (8). Others have described additional Smad-independent pathways. Bakin and colleagues reported that PI-3 kinase-Akt signaling was required for transcriptional activation by TGF- (37). Furthermore, related work by Bhowmick and coworkers has shown that p38 mitogen-activated protein kinase (MAPK) is activated independently of TGF- type II receptor activation, while biologic responsiveness also requires the transcriptional activation pathways of PI3-K and Smad (38). There is also some evidence that phosphorylation of a Smad adaptor protein can modulate Smad activation (39), but the participation of the adaptor in the process of mRNA stabilization is yet to be shown.

What is happening in the cell in the 4-6 h between the time when TGF- receptors become occupied and signal through Smads (a matter of minutes) and the observed effects on elastin mRNA levels and stability? What are the secondary mediators of TGF- in the sequence of events leading to elastin mRNA stabilization? The fact that cycloheximide inhibits the TGF- effect on elastin mRNA stability in lung fibroblasts implies that there is at least one round of transcriptional activation (presumably through Smads and their cofactors) and translation to produce an early response protein. After transfection with a Smad7 vector, Kucich and coworkers observed a residual TGF- effect (Figure 1 in Ref. 8). This could represent a Smad-independent component of signaling, but overexpression of Smad7 may not have reached 100% efficiency. In an alternate scheme to that proposed by Kucich and coworkers, TGF- activation of cells through Smad3/4 would stimulate the synthesis of autocrine/intracrine factor(s), whose ensuing signaling then activated pathways dependent on phospholipase and kinase activity.

TGF- modulation of cell-matrix interactions and cell shape could also produce independent, secondary, or tertiary responses that act through kinase pathways. TGF- has major effects on the upregulation of a host of matrix structural proteins. At the same time, matrix remodeling is inhibited by the suppression of matrix metalloproteinase transcription and the activated transcription of proteinase inhibitors such as PAI-1. Integrin receptors and the expression of cytoskeletal proteins such as -smooth muscle actin are also affected. Indeed, blockade of TGF- action by transfection of Smad 7 or dominant negative Smad 3 vectors alters transcriptional and growth responses in epithelial cells, but it has no effect on the rapid, RhoA-mediated reorganization of the cytoskeleton (43). In transformed epithelial cells, p38 kinase activity is also independent of Smad signaling, and it is necessary but not sufficient for morphologic changes (38).

Another form of downstream TGF- signaling is the expression of connective tissue growth factor (CTGF) (40). CTGF contains TGF--responsive elements (41, 42), and it has been proposed that a number of the effects on extracellular matrix attributed to TGF- are mediated through CTGF (40). Evidence from many laboratories shows that expression of this secreted growth factor is activated by TGF- within less than 2 h of TGF- exposure. Kucich and coworkers recently showed that CTGF induction requires a phosphatidyl-specific phospholipase-C, a protein kinase C, and one or more tyrosine kinases (9). It is not known to what extent this induction would affect mRNA stabilization, but CTGF still serves as a prototype for primary TGF-/Smad targets that could secondarily modulate processes such as elastin synthesis.

Dissection of the Smad signal pathway and its consequences has been facilitated by selective disruption of Smad elements. An important insight into those genes directly activated by the TGF-/Smad3 pathway comes from the work of Verrecchia and colleagues (44). These investigators used a combination of stringent criteria for direct Smad 3 activation that included speed (< 30 min), inhibition by both Smad 7 and dominant negative Smad 3 transgenes, activation by Smad 3 transfection, and lack of activity in fibroblasts from the Smad 3(/) mouse strain. Among the direct targets these authors identified by array screening were COL1A1, COL1A2, TIMP-1, and type V and type VI genes. In addition to cellular poisons, the use of Smad 3 (/) cells and dominant negative forms of the TGFRII, RhoA, and Smad 3 would help iron out the details of the signal pathway leading from TGF-R activation to elastin mRNA stabilization.

	Footnotes

Address correspondence to: Jeffrey M. Davidson, Ph.D., Department of Pathology, C-3321 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232-2561. E-mail: jeff.davidson{at}vanderbilt.edu

(Received in original form December 26, 2001).

Acknowledgments: This work was supported in part by NIH grant AG06528 and by the Department of Veterans Affairs.

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