BACKGROUND & AIMS Fibrosis is an abnormal extension of the wound healing process that follows tissue damage; it is involved in pathogenesis in a variety of chronic diseases. The formation of extracellular matrix is an essential response in wound healing. Although it has been proposed that collagen organization and assembly depend on the fibronectin matrix in culture, the contribution of fibronectin to these processes remains to be defined in vivo. METHODS We generated a conditional, fibronectin-deficient mouse model of liver injury and explored whether fibronectin would be a suitable target for preventing extensive collagen deposits and scar formation that could lead to liver fibrosis. RESULTS The lack of fibronectin did not interfere with reconstruction of collagen fibril organization in response to liver injury. Signaling by transforming growth factor (TGF)-β and type V collagen were required for collagen fibrillogenesis during remodeling of adult liver tissue. CONCLUSIONS TGF-β and type V collagen are targets for regulating the initial fibrogenic response to liver damage.
A full-length cDNA of smooth muscle regulatory light chain was obtained and the recombinant regulatory light chain was expressed in an Escherichia coli expression system. The recombinant regulatory light chain was introduced into myosin or HMM using a subunit exchange strategy [Morita, J., Takashi, R., & Ikebe, M. (1991) Biochemistry 30, 9539-9545]. The recombinant wild-type regulatory light chain exhibited the same biological properties as the natural isolate, i.e., phosphorylation at Ser-19 by myosin light-chain kinase and phosphorylation-activated actomyosin ATPase activity. To clarify whether or not the activation of the ATPase by phosphorylation is simply due to the introduction of negative charge, we produced three mutant light chains. Two of them contain Ser-19 substituted by either Asp or Ala and the third contains Asp substituted for both Thr-18 and Ser-19. Incorporation of the Asp mutant partially activated actomyosin ATPase activity but the activation level was significantly lower than that by phosphorylation. The Asp/Asp mutant further activated actomyosin ATPase activity. On the other hand, the Ala mutant did not affect the ATPase activity. Incorporation of Asp mutant slightly affected the 10S-6S conformational transition and filament formation of myosin. The Asp/Asp mutant more significantly affected the 10S-6S conformational transition and filament formation of myosin. These results suggested that the activation of smooth muscle myosin requires the introduction of negative charge in the defined spacial position. Using Ser-19 deficient mutants, the effects of Thr-18 phosphorylation on myosin function was also studied. Actin-activated ATPase activity of myosin was significantly activated by phosphorylation of Thr-18.(ABSTRACT TRUNCATED AT 250 WORDS)
The segment of smooth muscle regulatory light chain essential for the phosphorylation dependent activation of actomyosin motor activity and the binding of myosin heavy chain was identified. The C-terminal domain of the 20-kDa light chain, which is less conserved than the rest of the polypeptide among various muscle types, was mutated by either deletion or substitution of amino acid residues and the mutant light chains were then incorporated into myosin by subunit exchange. Deletion of Lys49-Ala'66 markedly reduced the affinity of the light chain for the heavy chain, whereas the C-terminal five residues, Lys167-Asp'71, did not contribute to the binding affinity. Deletion of Lysl49-Phelsw abolished the phosphorylation-dependent activation of actomyosin ATPase activity as well as superprecipitation activity. These results suggest that the C-terminal domain ofthe regulatory light chain is critical for transmitting the change in the conformation of the regulatory light chain induced by phosphorylation at Ser'9 to the heavy chain.The actomyosin function from vertebrate smooth muscle and nonmuscle cells is regulated by phosphorylation of the 20-kDa light chain (LC20) at Ser19 by Ca2+/calmodulindependent myosin light-chain kinase (1-3). LC20 is associated with the myosin heavy chain at the head-rod junction (4, 5) placing the phosphorylation site distally to both the catalytic and actin binding sites, which are located toward the top of the myosin head (4). The question, then, is how does the phosphorylation of LC20 at the specific site (Ser19/Thr'8) interact with the distal effector sites (ATP binding and actin binding sites) so as to activate the actin-activated myosin ATPase activity. The following findings suggest that the conformation at the head-rodjunction of myosin is altered by phosphorylation, presumably by a change in the interaction between LC20 and heavy chain, and that this is an important factor for the activation mechanism. (i) Heavy meromyosin, but not myosin subfragment 1 (S-1), containing intact regulatory light chain shows phosphorylation dependence of its actin-activated ATPase activity (6). (ii) The change in the head-rod junction conformation induced by LC20 phosphorylation can be detected by changes in susceptibility to proteases (7,8), changes in flexibility (9), and changes in shape observed by electron microscopy (10,11).It is plausible that the phosphorylation alters the conformation of LC20 and that this information is communicated to the heavy chain via LC20 heavy-chain binding sites. Skeletal muscle myosin can also be phosphorylated at the homologous site of LC20, but in this case, phosphorylation does not affect the ATPase activity of actomyosin. The difference is at least partly due to the differences in these regulatory light chains. This is underscored by the observations that, whereas smooth muscle regulatory light chain restores the Ca2+
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