- Top of page
- Materials and Methods
- Supporting Information
The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts.
Myofibroblasts are specialized contractile fibroblasts that are proposed to play a key role in generating contractile forces responsible for wound closure and pathological contractures.[1-4] These cells are characterized by the acquisition of a contractile phenotype, which includes the formation of large stress fibers and supermature focal adhesions.[5-7] In addition, myofibroblasts express smooth muscle α-actin (SMαA),[3, 8] an actin isoform found predominantly in smooth muscle cells. One of the key questions concerning myofibroblast formation and function is the role of SMαA in the acquisition of the contractile phenotype and the generation of contractile force.
There are six actin isoforms found in all mammalian cells: two cytoplasmic actin isoforms that are ubiquitously and highly expressed in nonmuscle cells, cytoplasmic β-actin (CYβA) and cytoplasmic γ-actin (CYγA), and four muscle actin isoforms that are named for their primary localization—SMαA, smooth muscle γ-actin (SMγA), skeletal muscle α-actin (SkMαA), and cardiac muscle α-actin (CMαA). SMαA makes up approximately 20% of the total actin found in myofibroblasts. Expression of SMαA in myofibroblasts has been correlated with the acquisition of the contractile phenotype and force generation.[3, 11] In addition, increased expression of SMαA by itself is sufficient to increase stress fiber and focal adhesion assembly and increase generation of contractile force. These results suggest that the expression of SMαA in myofibroblasts plays a key role in their formation and function. However, studies have showed that myofibroblasts express other smooth muscle contractile proteins which may also play an important role in myofibroblast formation and function, including SM22α, h1-calponin, and SMγA.[12, 13] Recent studies have showed that decreased expression of contractile genes with CArG elements in their promoter, including SMαA, SMγA, SM22α, and h1-calponin, can reduce stress fiber and focal adhesion assembly, as well as myofibroblast formation and function.[13, 14] These results raise the question as to whether SMαA is necessary for myofibroblast formation and function or whether other contractile proteins could compensate for SMαA.
Previous studies have shown that smooth muscle cells can still function in the absence of SMαA. SMαA-null mice are healthy and survive through adulthood, showing that both vascular and visceral smooth muscles can function without SMαA, although contractile force generation is reduced in both vascular and bladder smooth muscles of SMαA-null mice.[15, 16] The expression of other actin isoforms in the smooth muscle of these SMαA-null mice—SkMαA in vascular smooth muscle cells and SMγA in bladder smooth muscle cells—suggests that expression of these other actin isoforms may compensate for lack of SMαA. Interestingly, myoepithelial cell function is dramatically decreased in SMαA-null mice, suggesting that these epithelial-derived contractile cells cannot compensate due to the lack of expression of other muscle actin isoforms.[17, 18]
To determine the role of SMαA in myofibroblast formation and function during wound closure, we examined closure of excisional wounds on the dorsum of SMαA-null mice. In addition, SMαA-null fibroblasts were treated with transforming growth factor-β1 (TGF-β1), which promotes myofibroblast formation,[11, 19] and examined for their ability to acquire the myofibroblast phenotype and generate contractile force in tissue culture models of wound contraction. We found that SMαA is not necessary for excisional wound closure and that the mechanical and growth factor environment in SMαA-null wounds is sufficient to induce SMαA promoter activity. Fibroblasts in SMαA-null granulation tissue positively stained with a monoclonal antibody that recognizes all muscle actin isoforms, exhibiting a myofibroblast-like distribution and a stress fiber-like pattern, thus demonstrating that these cells acquired the myofibroblast phenotype. In addition, cultured SMαA-null fibroblasts can acquire the myofibroblast phenotype and generate contractile force similar to wild-type (WT) fibroblasts in response to TGF-β1. We have also demonstrated by immunostaining, real-time polymerase chain reaction (PCR), and mass spectrometry that SMγA and SKMαA are expressed in cultured SMαA-null myofibroblasts and organized into stress fibers. These results suggest that SMαA is not necessary for myofibroblast formation and function, and that other muscle actin isoforms and/or contractile proteins can compensate for its loss.
- Top of page
- Materials and Methods
- Supporting Information
In this paper, we demonstrate that mice lacking SMαA can close excisional wounds and that the mechanical and growth factor environments present in the granulation tissue of these SMαA-null mice are sufficient to promote activation of the SMαA promoter. In addition, we demonstrate that mouse dermal fibroblasts lacking SMαA can form stress fibers and focal adhesions and can generate increased contractile force in response to TGF-β1, similar to WT fibroblasts. There was a significant delay early in the closure of 4 mm wounds in SMαA-null mice; however, this delay disappeared by 4 days after wounding. It is unclear why lack of SMαA expression should delay early wound closure, as SMαA-positive myofibroblasts appear later during wound closure.[3, 33] Previous studies have showed a correlation between SMαA-positive myofibroblasts and tissue contraction, suggesting a role for SMαA in generating cellular contractile force.[3, 8, 34] In addition, increased expression of SMαA will result in increased formation of stress fibers and focal adhesions and in increased contractile force generation.[3, 11] Therefore, at first glance it is surprising that SMαA-null wounds closed and SMαA-null fibroblasts could acquire the myofibroblast contractile phenotype and force generation; however, a number of studies on other cell types have showed that other muscle actin isoforms can, at least partially, compensate for the lack of a muscle actin isoform.[15, 16, 35, 36] These results would suggest that myofibroblasts have a mechanism for compensating for the lack of expression of SMαA, perhaps by expression of other muscle actin isoforms.
Fibroblasts in SMαA-null granulation tissue were found to express muscle actin isoforms other than SMαA, as evidenced by immunostaining with clone B4, and organize these muscle actin isoforms into stress fiber-like structures, suggesting that other muscle actin isoforms are expressed and can potentially compensate for the lack of SMαA. Cultured SMαA-null mouse dermal fibroblasts treated with TGF-β1 to promote myofibroblast formation were found to express both SMγA and SkMαA. The loss of SMαA in myofibroblasts resulted in no decrease in total actin levels and no increase in CYβA or CYγA, showing that the expression of these two cytoplasmic actin isoforms did not increase to compensate for the loss of SMαA in myofibroblasts. SMγA was expressed in SMαA-null myofibroblasts as shown by immunostaining with clone CGA7 and real-time RT-PCR. Because clone CGA7 recognizes SMαA, in addition to SMγA, it was not possible to determine whether SMγA protein is increased in SMαA-null compared with WT myofibroblasts; however, SMγA mRNA is significantly increased in SMαA-null compared with WT myofibroblasts, suggesting that SMγA is increased in response to lack of SMαA. SkMαA was observed to increase in SMαA-null compared with WT myofibroblasts both by immunostaining with clone 5C5 and by RT-PCR. The level of expression of SMγA and SkMαA in SMαA-null myofibroblasts appears to be significantly less than the expression of SMαA in WT myofibroblasts; whether the expression of SMγA and SkMαA occurs in the same cell, reaching the level of SMαA expression, is unclear. While we have demonstrated that SMγA and SkMαA are expressed and organized into stress fibers in SMαA-null myofibroblasts, further studies are necessary to determine the relative level of expression of these actin isoforms to each other and to the expression of SMαA in WT myofibroblasts.
The mechanism by which SMγA and SkMαA may functionally compensate for the loss of SMαA is currently unknown. Consistent with our findings, Chambers and co-workers have demonstrated that human fetal lung fibroblasts treated with TGF-β1 increase SMαA, SMγA, and CMαA expression. We did not see expression of CMαA in myofibroblasts, suggesting that myofibroblasts from different locations may express different muscle actin isoforms in response to TGF-β1. The amino acid sequences for the six actin isoforms are conserved for vertebrates, suggesting strong evolutionary pressure to maintain these specific sequences and differential function for each isoform. Currently, the function for the different actin isoforms is not completely understood. Studies have suggested that in nonmuscle cells the two cytoplasmic actin isoforms, CYβA and CYγA, have different localizations and different functions.[5, 37, 38] Several lines of evidence suggest that other muscle actin isoforms can functionally compensate, at least partially, in CMαA-null mice and SkMαA-null mice. Similarly, SMαA-null mice have no major phenotype and SkMαA has been proposed to functionally compensate, at least partially, in vascular smooth muscle cells. Myoepithelial cell function is dramatically decreased in SMαA-null mice such that lactating females cannot nurse their pups.[17, 18] This inability for compensation may be due to lack of expression of other muscle actin isoforms in these epithelial-derived contractile cells. Interestingly, human genetic disorders have recently been described with mutations in SMαA which cause cardiovascular defects; in this case, compensation may not be possible as these mutations appear to be dominant negative. Myofibroblast contractile function has been shown to be perturbed by the peptide, Ac-EEED, homologous to the N-terminal sequence of SMαA. These results suggest that the acute perturbation of SMαA function may not allow time for compensation mechanisms to come into play, as can occur in null animals. Consistent with this, acute knockdown of SMαA using antisense oligonucleotides decreased focal adhesion formation and increased cell migration. Recent work has shown that there is a broad genetic reprogramming of β-actin-null cells that does not occur in acute knockdown of β-actin. It may be that whole animal knockout of SMαA results in a genetic reprogramming that allows for compensation that does not occur with acute knockdown or loss of function of SMαA. Further experiments comparing SMαA-null with acute SMαA knockdown in fibroblasts will be needed to determine the mechanisms for compensation and possible genetic reprogramming.
In cardiac and smooth muscle cells, the expression of muscle actin isoforms and other contractile proteins is regulated by the transcription factor, myocardin, through its binding to serum response factor and to CArG elements in the promoter of these genes. Nonmuscle cells do not express myocardin; rather, they express the related transcription factors myocardin-related transcription factor A and B (MRTF-A, MRTF-B). We have recently demonstrated that knockdown of MRTF-A and -B in myofibroblasts will significantly reduce expression of SMαA and SMγA and other contractile proteins, and significantly reduce the myofibroblast contractile phenotype and its ability to generate contractile force. These results are consistent with the results presented in the current study, in that loss of expression of SMαA can be compensated for, and that knockdown of all muscle actin isoforms and other contractile proteins is required to reduce myofibroblast formation and function.
TGF-β1 treatment of SMαA-null fibroblasts significantly increases SkMαA mRNA, increases SMγA mRNA, although not significantly, and increases significantly mRNA for SM22α and h1-calponin (Tomasek, unpublished results), all of which may play a role in promoting myofibroblast formation and function in the absence of SMαA. The mechanism by which TGF-β1 may promote increased expression of SkMαA and SMαA is currently unclear. We have previously proposed that TGF-β1 may promote expression of myofibroblast contractile genes by directly altering actin dynamics through a Rho-mediated mechanism and thereby promote nuclear translocation of MRTF-A/B. SMγA and SKMαA promoters have CArG elements that bind SRF that may be regulated by MRTF-A/B.[43-45] However, TGF-β1 has also been shown to increase expression of SMαA and SM22α in fibroblasts through TGF-β1 control elements[46-48] and/or Smad-binding elements.[49, 50] Further studies will be needed to understand how TGF-β1 promotes increased expression of these contractile proteins and their role in promoting myofibroblast formation and function in SMαA-null fibroblasts.
We have demonstrated that loss of SMαA in myofibroblasts results in increased expression of SMγA and SkMαA, and that the expression of these muscle actin isoforms may functionally compensate for the loss of SMαA. Myofibroblasts express, in addition to muscle actin isoforms, other contractile proteins such as SM22α and h1-calponin, which also appear to regulate myofibroblast formation and function. To understand myofibroblast formation and function, it will be necessary to identify the multiple contractile proteins expressed, to understand how their expression is regulated, and to determine how they interact to promote the contractile phenotype and force generation so important in wound closure and pathological contractures.