In the present study, we examined the contribution of p70S6K to cellular hypertrophy. We found that treatment with TGF-β, ET-1, and CT-1, each of which have been implicated in asthmatic airway remodeling and the induction of airway smooth muscle hypertrophy (4
), increased phosphorylation of both p70S6K and its downstream target ribosomal protein S6. Further, TGF-β–, ET-1–, and CT-1–mediated increases in airway smooth muscle cell size and protein synthesis were blocked by specific siRNA against p70S6K and S6, as well as overexpression of kinase-dead p70S6K. However, TGF-β–, ET-1–, and CT-1–induced increases in α-smooth muscle actin, MHC and SM22 expression relative to β-actin were not affected by these interventions. Moreover, overexpression of a constitutively active form of p70S6K elicited an increase in airway smooth muscle cell size but not contractile protein expression or cell shortening. Together, these data suggest that activation of p70S6K is required and sufficient for myocyte enlargement in cultured airway smooth muscle cells. Further, they demonstrate that p70S6K-mediated cell size enlargement is due to a global increase in cellular protein, including cytoskeletal proteins such as β-actin, rather than a preferential increase in contractile proteins. Finally, these data show for the first time in a functional cell system that myocyte enlargement is insufficient for increased cell shortening.
As noted above, TGF-β, ET-1, and CT-1 have each been noted to induce elements of hypertrophy of cultured airway smooth muscle cells. TGF-β augments cell size and contractile protein synthesis through several distinct mechanisms. TGF-β treatment induces phosphorylation of eukaryotic translation initiation factor-4E–binding protein (8
) and phosphorylation of GSK-3β (14
). Phosphorylation and inhibition of GSK-3β, in turn, derepresses transactivation of nuclear factors of activated T cells (NFAT), and serum response factor, transcription factors involved in muscle-specific gene expression. Inhibition of GSK-3β also reduces phosphorylation of eukaryotic initiation factor-2B
, thereby promoting translation of contractile protein–encoding mRNAs. ET-1 increases airway smooth muscle protein synthesis and α-actin, SM22, and calponin expression in an extracellular signal regulated kinase–dependent manner (13
). CT-1 increases airway smooth muscle cell size, protein synthesis (9
), contractile protein expression, and GSK-3β phosphorylation (14
). Finally, inhibition of the GSK-3β pathway is required for CT-1– but not TGF-β–induced hypertrophy (14
The precise contribution of p70S6K activation to airway smooth muscle hypertrophy has not been established. Further, the functional consequences of p70S6K-mediated hypertrophy have not been studied. In C2C12 mouse skeletal myoblasts, selective activation of p70S6K was sufficient for myotube hypertrophy (increased cell size) (25
). Rapamycin, a chemical inhibitor of mTOR, fails to attenuate myotube formation or myosin heavy chain expression in rat L6E9, mouse Sol8 and primary human myoblasts (26
). Cardiac-specific transgenic mice overexpressing a rapamycin-resistant mutant of p70S6K with higher basal activity show cardiac hypertrophy, as assessed by heart weight, though deletion of p70S6K does not attenuate insulin-like growth factor-I or phosphatidylinositol 3-kinase–mediated cardiac hypertrophy (27
). The effects of RR p70S6K expression on cardiac function were not examined. In vascular smooth muscle, chemical inhibitors of p70S6K (tosylphenylalanine chloromethyl ketone and tosyllysine chloromethyl ketone) had no effect on angiotensin II–induced protein synthesis (28
). Together, these results suggest that p70S6K is sufficient but not required for muscle hypertrophy.
In the present study, using conditions optimized for the induction of cellular hypertrophy, we obtained different results. First, we found that inhibition of p70S6K with either specific siRNA or kinase-dead p70S6K blocked TGF-β–, ET-1–, and CT-1–induced airway smooth muscle protein synthesis and cell enlargement, whereas expression of rapamycin-resistant p70S6K was sufficient to induce these effects, demonstrating that p70S6K is both required and sufficient for airway smooth muscle cell enlargement. Second, when we examined the full range of hypertrophic responses, a more complex picture emerged. Neither specific siRNA nor KD blocked expression of α-smooth muscle actin, MHC, or SM22 relative to β-actin. Moreover, RR p70S6K failed to selectively increase contractile protein expression. When contractile responses to KCl were examined, expression of KD p70S6K failed to inhibit TGF-β–induced shortening. Finally, longer RR p70S6K cells failed to shorten in response to KCl in the absence of TGF-β. Together, these data suggest that increases in cell size alone, without concurrent preferential increases in contractile protein expression, are insufficient to alter airway smooth muscle function. The failure of p70S6K to regulate contractile protein expression is analogous to recent results we have obtained in canine tracheal smooth muscle cells expressing a constitutively-active form of the serine-threonine kinase Akt1 (L. Ma, J. Solway, unpublished data). Akt activation induced cellular enlargement without increasing expression of α-smooth muscle actin or SM22. Together, these studies hold relevance for the functional significance of airway smooth muscle remodeling in asthma. While increased smooth muscle mass is the most prominent pathologic change observed in the airways of patients with asthma, it is unclear whether cell enlargement is accompanied by specific increases in contractile protein expression. There are relatively few studies examining smooth muscle contractile protein expression in asthma. Two reports showed increased myosin light chain kinase expression (2
), while another study showed no increase in the mRNA expression of genes encoding contractile proteins (3
). It also remains unclear whether hypertrophy is sufficient to induce a state of airways hyperresponsiveness, the physiological hallmark of asthma. In the present study, we show for the first time that enlarged airway smooth muscle cells do not develop increased shortening in the absence of concurrent changes in contractile protein expression.
The precise mechanism by which p70S6K controls translation is unclear. Other targets of p70S6K exist besides ribosomal protein S6, including the transcription factor cAMP-responsive element modulator (30
), the 80-kD subunit of the nuclear RNA cap-binding complex, CBP80 (31
), eukaryotic elongation factor-2 kinase (18
), and the pro-apoptotic protein Bcl-xL/Bcl-2-associated death promoter (BAD) (32
). p70S6K also mediates assembly of eukaryotic initiation factor-3 translation preinitiation complex (19
). In our study, we found discrepancies between the level of p70S6K and S6 phosphorylation following mediator stimulation, consistent with the notion that p70S6K has multiple downstream phosphorylation targets. However, we also found that phosphorylation of ribosomal protein S6 was increased in RR p70S6K cells and decreased in KD p70S6K cells, demonstrating that p70S6K regulates S6 phosphorylation. However, it is unclear from these data alone whether S6 is responsible for p706K-mediated airway smooth muscle hypertrophy. To test this, we examined the effects of S6 knockdown on myocyte size and contractile protein. Like p70S6K knockdown, siRNA directed against S6 blocked TGF-β–induced cell size enlargement. Further, S6 knockdown blocked RR p70S6K-mediated myocyte hypertrophy. Together these results suggest that, in airway smooth muscle, p70S6K-mediated hypertrophy is mediated by ribsosomal protein S6.
We would like to mention some potential limitations of our study. First, as noted in the Introduction, we studied cells in serum-free medium supplemented with TGF-β, ET-1, or CT-1 for 4 days to obtain a hypertrophic cell phenotype. Because hypertrophy depends in part on post-transcriptional mechanisms, and because the turnover of contractile proteins is relatively slow, the hypertrophic phenotype takes some days to develop. Under these conditions, it is possible that p70S6K was stimulated not only by these stimuli, but also by a second autocrine factor. Nevertheless, we felt that this experimental strategy was justified by our interest in the common downstream signal transduction mechanisms regulating hypertrophy, rather than the specific stimuli per se. On this basis, we also chose not to examine the specific concentrations of TGF-β, ET-1, and CT-1 necessary for the hypertrophic phenotype. Finally, in our study, we measured the shortening of unloaded dispersed airway smooth muscle cells to KCl stimulation. Measuring unloaded shortening capacity can be complicated by the possibility that the degree of activation and starting length may vary from cell to cell due to the preparation procedure. To address this, cells were treated with a cell permeable cyclic AMP analog, 8-bromo-cAMP, and allowed to “relax” overnight prior to study. Nevertheless, we acknowledge that results could be different in living, excised strips of airway smooth muscle.
In conclusion, TGF-β, ET-1, and CT-1 cause airway smooth muscle cell size enlargement and protein synthesis, which require activation of p70S6K. However, selective increases in contractile protein synthesis caused by these factors are not dependent on p70S6K. Further, activation of p70S6K is sufficient for cellular enlargement, but not for contractile protein expression. Finally, hypertrophic RR p70S6K-expressing cells fail to demonstrate enhanced shortening. On this basis, we speculate that increases in airway smooth muscle mass must be accompanied by preferential increases in contractile protein expression if they are to induce airways hyperresponsiveness in asthma. Further studies examining the biology of airway smooth muscle hypertrophy may provide insight into the pathogenesis and functional significance of airway remodeling.