INTRODUCTION

TOP ABSTRACT INTRODUCTION INTEGRIN FUNCTION IN SKELETAL... DOWNSTREAM OF INTEGRIN-MEDIATED... INTEGRIN SIGNALING AND SKELETAL... FUTURE DIRECTIONS REFERENCES

THE PAST 10 YEARS have seen a dramatic increase in our understanding of load-induced signaling that alters the mass and/or phenotype of skeletal muscle (3, 7, 14). A major focus of this increased knowledge has been the regulation of skeletal muscle protein synthesis. The rate of skeletal muscle protein synthesis is a critical regulatory event leading to load-induced changes in skeletal muscle mass (31, 39, 41, 72). Molecular biology approaches have made a large contribution to our current understanding of how mechanical loading can alter gene expression and muscle protein synthesis rates in skeletal muscle.

The use of transgenic mice (44, 45, 64, 65, 70) and plasmid DNA injection into skeletal muscle (11, 24, 60, 69) are examples of molecular biology approaches employed to study skeletal muscle mass and phenotype regulation. Cell-culture models have also been used to examine the effects of loading on gene regulation in skeletal muscle cells (15). These in vivo and in vitro approaches have established that mechanical forces can alter the regulation of skeletal muscle genes. Furthermore, specific regulatory regions of these genes have been identified that, at least partially, regulate the gene's response to mechanical forces.

Transcription factor proteins can bind regulatory regions of DNA and subsequently alter gene transcription. Specific transcription factor proteins have been implicated for mechanical load signaling in skeletal muscle (13, 45, 69). Phosphorylation signaling cascades can regulate transcription of a gene by altering a transcription factor's affinity for DNA, ability to form protein-protein interactions, and/or the activity of the protein. The linkage of load-activated signaling cascades with specific transcription factors and/or translation-related protein targets has not been firmly established in skeletal muscle. Exercise and overload have been shown to activate signaling cascades in skeletal muscle (2, 4, 28). However, these signaling cascades have not been directly linked with protein targets that alter skeletal muscle gene expression. Defining these signaling pathways with their target regulatory molecules that alter gene expression will provide much needed insights into our understanding of how mechanical loading regulates protein synthesis and, consequently, the regulation of skeletal muscle mass.

The scope of this review is the examination of evidence implicating integrin-mediated signaling as a candidate for influencing load-induced changes in skeletal muscle protein synthesis through the regulation of gene transcription and/or mRNA translation. Key components of integrin-mediated signaling that are candidates for linking mechanical signals at the muscle membrane to molecular events that regulate gene expression and, ultimately, muscle growth are identified. Basic integrin function and its role in cellular processes will be only briefly discussed, since a detailed review of this topic would necessitate its own book chapter. The reader is encouraged to examine several excellent reviews in this area for more detailed information (18, 30, 36, 56).

	INTEGRIN FUNCTION IN SKELETAL MUSCLE

TOP ABSTRACT INTRODUCTION INTEGRIN FUNCTION IN SKELETAL... DOWNSTREAM OF INTEGRIN-MEDIATED... INTEGRIN SIGNALING AND SKELETAL... FUTURE DIRECTIONS REFERENCES

The cellular functions of integrins have been extensively researched and are important for numerous cellular events including gene expression, cell growth, proliferation, differentiation, and apoptosis (18, 56). Integrins are thought to be a primary sensor for relaying physical or mechanical signals from the surrounding environment into the interior of the cell, which then allows for the appropriate cellular response (36). The integrin family's mechanical sensing function can be fulfilled, because the integrins are membrane-associated proteins involved in maintaining cell interactions with the extracellular matrix and/or other cells. Integrins maintain dual roles in cell adhesion and signal transduction. Thus integrins serve as a link between the extracellular matrix and the cell's cytoskeleton and constitute a critical component of the signaling process by which the cell integrates mechanical signals from its surrounding environment.

The integrin family can be described as heterodimeric transmembrane glycoproteins, which consist of - and -subunits. At least 16 - and 8 -integrin subunits have been characterized (18). There is also alternative splicing of the prospective mRNA's extracellular and cytoplasmic domains (18). Multiple subunit isoforms and alternative splicing give tremendous diversity to the integrin family. The combination of - and -subunits alters both integrin affinity for extracellular matrix proteins and the internal signaling cascades that are activated (30). Many integrins maintain a restricted expression pattern. The cytoplasmic domain of the -integrin subunit, rather than the -subunit, is primarily required for interaction with the cytoskeleton (45). A muscle-specific splice variant of the 1- subunit (1D) has been identified (26). The 1D-subunit is specifically expressed in skeletal and cardiac muscle, and represents an alteration in the cytoplasmic splicing of the 1-subunit (26). The integrin isoform shift to the 1D-isoform is essential for myoblast differentiation during development (53). It has not been documented whether mechanical load induces integrin splicing variants or a developmental program of integrin isoform expression in skeletal muscle.

Ligand binding to the integrin receptor causes integrin clustering, which can serve to activate tyrosine kinases (18). The specific ligand or extracellular matrix molecule associating with the integrin receptor can influence the specificity of the signaling (18). There is no intrinsic enzyme activity contained in the cytoplasmic domains of integrin subunits, and signaling is propagated by the formation of large protein complexes containing cytoskeletal and catalytic proteins (18). Activation of cellular signaling through extracellular matrix-integrin interactions initially occurs by the formation of focal adhesion complexes (9). Focal adhesion complex formation is necessary for linking integrins to the cytoskeleton and to other signal transduction proteins. The focal adhesion complex involves the accumulation of vinculin, talin, and -actinin proteins, which are necessary for linkage of integrins to the cytoskeleton (35). Focal adhesion complex formation appears to be sensitive to mechanical forces. The 1-subunit and vinculin are stabilized by the static stretch of cardiac myocytes through focal adhesion formation and maintenance (57). Indexes of focal adhesion complexes also increase in chronically stretched skeletal muscle (28). The formation of focal adhesion complexes and the subsequent initial tyrosine phosphorylation of signal transduction proteins is one of the earliest and most critical components for transduction of mechanical signaling. Focal adhesion complexes may also serve as a key integration site for multiple signals that are present during overload-induced skeletal muscle hypertrophy (28).

	DOWNSTREAM OF INTEGRIN-MEDIATED SIGNALING

TOP ABSTRACT INTRODUCTION INTEGRIN FUNCTION IN SKELETAL... DOWNSTREAM OF INTEGRIN-MEDIATED... INTEGRIN SIGNALING AND SKELETAL... FUTURE DIRECTIONS REFERENCES

Integrin interaction with the extracellular matrix and the subsequent formation of focal adhesion complexes can directly alter transcription (29) and translation (42). Integrator or target proteins involved in the integrin regulation of gene expression have been well studied, and many components have been identified in various cell types (9, 18, 55). A vast array of signaling molecules and cascades have been connected to integrin signaling, including focal adhesion kinase (FAK), protein kinase C, mitogen-activated protein kinase, phosphatidylinositol-3 kinase (PI-3 kinase), Ras, and Rho to name a few (18) (Fig. 1). The breadth of these signaling cascades is beyond the scope of this review. However, cascades that have been described during or are related to skeletal muscle growth are discussed.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Signaling pathways that can be activated in skeletal muscle by integrin-mediated signaling. An overload stimulus can potentially activate many different signaling cascades in skeletal muscle. However, how all these signaling pathways are integrated into a growth response is not completely understood. Integrin/RhoA signaling in skeletal muscle is an excellent candidate for integrating at least some of these events for the induction of hypertrophy. FAK, focal adhesion kinase; PI-3 kinase, phosphatidylinositol-3 kinase; JNK, c-Jun NH2-terminal kinase; PIP2, L--phosphatidylinositol 4,5-diphosphate.

Downstream Signaling Mediator: FAK

Downstream Signaling Mediator: RhoA

RhoA-mediated signaling has been shown to alter gene transcription (34). Activated RhoA induces the activity of several promoters including skeletal -actin, atrial natriuretic factor and c-fos (66, 67). Skeletal -actin and c-fos promoter activation by RhoA is dependent on serum-response factor (SRF) binding at a serum-response element (SRE) (66, 67). However, the signaling downstream of RhoA that alters gene transcription has not been clearly delineated. Several potential pathways could be involved, such as p38 kinase, c-Jun N-terminal kinase, and PI-3 kinase cascades, although their connection to SRF regulation is not certain (67). RhoA-SRF activation of the skeletal -actin promoter occurs through an integrin-dependent pathway (68). Overexpression of either 1D- or 1A-subunits can enhance RhoA activation of skeletal -actin and, conversely, a dominant negative mutant of 1-integrin inhibits RhoA activation (68). Downstream effector molecules of RhoA signaling have been implicated in cardiac hypertrophy. Rho kinase affects cardiac myocyte myofibrillar formation and is an important component of Rho-induced cardiac hypertrophy (35). p160ROCK is a mediator of endothelin-1 (ET-1) induction of the brain natriuretic peptide promoter by RhoA in hypertrophying cardiac myocytes (40). Although the mediator of p160ROCK signaling to the nucleus is not certain, inhibitors of c-Jun NH₂-terminal kinase can block ET-1-induced cardiac hypertrophy (17). The involvement of RhoA-dependent signaling, including downstream effector molecules, for regulating transcription and/or translation during skeletal muscle growth has not been established.

	INTEGRIN SIGNALING AND SKELETAL MUSCLE HYPERTROPHY

TOP ABSTRACT INTRODUCTION INTEGRIN FUNCTION IN SKELETAL... DOWNSTREAM OF INTEGRIN-MEDIATED... INTEGRIN SIGNALING AND SKELETAL... FUTURE DIRECTIONS REFERENCES

The function of integrin-mediated signaling has been extensively examined in cardiac muscle and linked to some types of cardiac hypertrophy (59). However, the ramifications of integrin signaling during changes in skeletal muscle mass are not well understood and need to be established. Cardiac and skeletal muscle load-induced regulation of gene expression is not always similar (15). Studies are needed to directly link integrin signaling to overload-induced skeletal muscle hypertrophy.

FAK is activated by the mechanical stretch of muscle (28, 57, 62). Interestingly, stretch activation of FAK and paxillin in smooth muscle is not dependent on tension development but only sensitive to muscle length changes (62). FAK and paxillin protein abundance increases in hypertrophying muscle (28). Chick and rat skeletal muscle subjected to chronic overload for as little as 24-36 h increases the protein concentration of FAK (28). FAK activity is also increased in hypertrophying chick skeletal muscle and is independent of increased FAK protein concentration (28). These observations suggest an increase in focal adhesion complex formation in hypertrophying skeletal muscle. Further work is needed to delineate whether the activation/induction of FAK in stretched skeletal muscle occurs through integrin-dependent signaling. An important remaining issue is the identification of FAK targets and functions in hypertrophying skeletal muscle.

Stretching the avian anterior latissimus dorsi (ALD) muscle increases muscle fiber length, fiber cross-sectional area, fiber number, and satellite cell activity (1, 71). Satellite cell activation is increased after 24 h of stretch, and peaks during the first week (12, 71). FAK activity can regulate skeletal myoblast proliferation and differentiation (53). Increased FAK protein abundance appears to correlate with satellite cell activation rather than increased fiber area or number. ALD muscle fiber area is not increased until day 7, and fiber number is not elevated until day 5 of stretch overload (1). This also appears true for increased FAK protein in rat hindlimb muscle during the first week of compensatory hypertrophy (28). Irradiated skeletal muscle might provide insight into FAK's role regarding satellite cell activity in skeletal muscle.

It is likely that increased FAK protein also has an important function for the hypertrophy response of preexisting fibers to overload. The ALD initially increases muscle fiber length 30-50% at the onset of stretch overload, which could serve to induce an increase and/or reorganization of focal adhesion complexes (1). A strong chronic stretch is also placed on rat plantar flexor muscles after synergist ablation due to a change in the agonist-to-antagonist muscle ratio. Stretch induces the addition of sarcomeres both in series and in parallel in both preexisting and newly formed muscle fibers. There is the possibility that the abundance of FAK protein within enlarging muscle fibers, compared with satellite and other mitotically active cells, would make up a larger percentage of total FAK protein as hypertrophy continues. It is also intriguing to consider that within enlarging preexisting muscle fibers increased FAK activity, not FAK abundance, may be the more important growth regulator. Further work will provide valuable insight into these important findings in hypertrophying skeletal muscle.

Calcium signaling, through a calcineurin-dependent pathway, can control the phenotype of skeletal muscle (16) and has also been reported necessary for compensatory hypertrophy of the rat plantaris muscle (23). Integrins have been shown to regulate intracellular calcium levels in some cell types (47). However, stretch-induced activation of FAK and paxillin occurs independently of intracellular calcium in smooth muscle (62). Initially, calcineurin signaling was reported essential for overload-induced hypertrophy of cardiac muscle (48). However, subsequent studies have demonstrated that overload-induced hypertrophy of rodent cardiac muscle can occur when the calcineurin pathway is blocked (22, 47, 73) and strongly suggest calcineurin- dependent and -independent pathways for cardiac hypertrophy. Further work is needed to determine whether calcineurin-dependent and -independent pathways are present in overloaded skeletal muscle. Satellite cell activation has been shown to be essential for compensatory hypertrophy of rodent skeletal muscle (50). It has not been determined whether calcineurin inhibitors diminish satellite cell proliferation and/or differentiation during compensatory hypertrophy. The determination of whether integrin-mediated signaling and/or FAK activation occur through calcineurin-dependent or -independent pathways in overloaded skeletal muscle will improve our understanding of this mechanism.

There is strong indirect evidence for examining integrin-mediated signaling during skeletal muscle hypertrophy. RhoA-integrin signaling mechanisms that alter gene expression during development of striated muscle have been linked to the transcription factor protein SRF (10, 66, 67). SRF is a phosphoprotein with a highly conserved DNA-binding and dimerization domain that has homology with the MADS box family of DNA-binding proteins including MEF-2, and whose concentration is enriched in muscle (20). SRF is essential for transcriptional regulation of many genes including the actin family and c-fos (8, 42, 63).

SRF is a mediator of both growth factor and mechanical signaling (15, 51, 62). There is the possibility that RhoA could integrate stimuli regulating transcription/translation during overload-induced skeletal muscle growth. The synergism of growth factor and integrin signaling through RhoA has been hypothesized to occur through RhoA's regulation of substrate availability for growth factor receptor pathways (46). The stage model of overload-induced skeletal muscle growth suggests that during the time course of growth there is a shift from mainly translational protein synthesis regulation to include more transcriptional control (14) (Fig. 2). This shift in skeletal muscle regulation could occur as local growth factor expression increases during the first week of stretch overload. Additionally, mechanical stretch and growth factors can interact to influence skeletal -actin promoter regulation in cultured myocytes (15). RhoA is an excellent candidate to mediate at least some part of this process.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Several levels of cellular regulation appear critical for large overload-induced increases in skeletal muscle mass. These points of regulation may have varying degrees of importance for inducing protein synthesis during the time course of overload-induced growth, especially in animal models of extreme hypertrophy. It is critically important to define how different cellular events are integrated to allow for a sequential growth response in skeletal muscle. eIFs, eukaryotic initiation factors.

SRF appears to be an important target for altered gene regulation in hypertrophying skeletal muscle by mechanical signaling. Skeletal -actin promoter activity is increased during the first week of chronic stretch overload in the chick ALD muscle (11). Stretch overload-induced activation of the skeletal -actin promoter requires SRF interaction with SRE 1 (13). SRF protein and mRNA abundance are increased in hypertrophying chick muscle, and the induction is correlated with most rapid periods of stretch-induced growth (28). RhoA activates the skeletal -actin promoter through SRF by an integrin-dependent mechanism in cell culture (67, 68). Further delineation of the relationship between integrins-RhoA-FAK and SRF, and identification of other possible rate-limiting components of this pathway will provide a stronger linkage and better understanding of overload-induced skeletal muscle hypertrophy. A component of mechanical overload signaling that would integrate diverse stimuli (i.e., stretch, growth factors) into an organized regulated response inducing growth has not been established in skeletal muscle. However, SRF does integrate stretch and serum signaling at the level of the promoter in cultured skeletal myotubes (15).

	FUTURE DIRECTIONS

TOP ABSTRACT INTRODUCTION INTEGRIN FUNCTION IN SKELETAL... DOWNSTREAM OF INTEGRIN-MEDIATED... INTEGRIN SIGNALING AND SKELETAL... FUTURE DIRECTIONS REFERENCES

The identification of molecular switches that integrate responses will be an important key for unlocking a better understanding of a complex phenomenon like overload-induced muscle growth (Figs. 1 and 2). Work has progressed to the point that several laboratories have now demonstrated how specific target genes are regulated during overload-induced skeletal muscle hypertrophy. Researchers have even identified specific transcription regulatory proteins that appear important for the response. Additional work has identified signaling pathways activated in hypertrophying muscle. However, what remains elusive is the mechanism by which all these genes and signaling pathways work together to give a unified response resulting in muscle enlargement and/or an altered phenotype due to loading demands. A more global view of cellular regulation is necessary for an understanding that will lead to new paradigms of specific and effective countermeasures for regulating skeletal muscle mass while minimizing or ablating deleterious side effects. This perspective will not only enhance our knowledge of the basic skeletal muscle biology but also provide the basis for effective therapeutic countermeasures and pharmacological interventions to treat a myriad of degenerative muscle conditions. These conditions could include individuals with acquired immune deficiency wasting syndrome, sarcopena, and severe arthritic conditions, to name just a few. New technologies such as microarray analysis should pave the way for the study of overload-induced skeletal muscle hypertrophy as a system of regulation, rather than the examination of the separate individual components, as is currently done. The near future should begin to provide insights into "the big picture" of regulation involved in the process of overload-induced skeletal muscle enlargement.