ABSTRACT
The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/mum(2)) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38alpha was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect beta-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-beta-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5-3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation.
Subject(s)
Actins/physiology , Biomechanical Phenomena , Muscle, Smooth/chemistry , p38 Mitogen-Activated Protein Kinases/metabolism , Actins/genetics , Actins/metabolism , Animals , Cell Line , Integrin alpha2beta1/metabolism , Muscle, Smooth/cytology , Phosphorylation , Protein Binding , Protein Isoforms , Rats , Receptors, Collagen , rho GTP-Binding Proteins/metabolismABSTRACT
To identify nucleotides in or near the active site, we have used a circularly permuted version of the VS ribozyme capable of cleavage and ligation to incorporate a single photoactive nucleotide analog, 4-thio- uridine, immediately downstream of the scissile bond. Exposure to UV light produced two cross-linked RNAs, in which the 4-thio-uridine was cross-linked to A756 in the 730 loop of helix VI. The cross-links formed only under conditions that support catalytic activity, suggesting that they reflect functionally relevant conformations of the RNA. One of the cross-linked RNAs contains a lariat, indicative of intramolecular cross-linking in the ligated RNA; the other is a branched molecule in which the scissile phosphodiester bond is cleaved, but occupies the same site in the ribozyme-substrate complex. These are the two forms of the RNA expected to be the ground state structures on either side of the transition state. This localization of the active site is consistent with previous mutational, biochemical and biophysical data, and provides direct evidence that the cleavage site in helix I interacts with the 730 loop in helix VI.
Subject(s)
Cross-Linking Reagents/pharmacology , Endoribonucleases/chemistry , Fungal Proteins/chemistry , Neurospora crassa/enzymology , RNA, Catalytic/chemistry , RNA, Fungal/chemistry , Thiouridine/pharmacology , Base Sequence , Binding Sites , Catalytic Domain , Endoribonucleases/drug effects , Endoribonucleases/radiation effects , Fungal Proteins/drug effects , Fungal Proteins/radiation effects , Molecular Sequence Data , Nucleic Acid Conformation , Photochemistry , RNA, Catalytic/drug effects , RNA, Catalytic/radiation effects , RNA, Fungal/drug effects , RNA, Fungal/radiation effects , Ultraviolet RaysABSTRACT
We examined mechanotranscriptional regulation of the contractile gene, alpha-smooth muscle actin (SMA), in osteoblastic cells. Tensile forces were applied through collagen-coated magnetite beads to ROS17/2.8 cells. These cells were desmin-, vimentin+ and expressed low levels of SMA. After force application (480 piconewton/cell), SMA protein and mRNA were increased but beta-actin was unchanged. Beads coated with bovine serum albumin or poly-L-lysine produced no change of SMA. In cells transiently transfected with plasmids containing the SMA promoter fused to beta-galactosidase or green fluorescent protein coding sequences, SMA promoter activity was increased by approximately 60% after 4 h of force, whereas control (Rous sarcoma virus) promoter activity was unaffected. Transfections with beta-galactosidase or green fluorescent protein reporter constructs showed that force-loaded cells exhibited higher beta-galactosidase activity than cells without force. Cytochalasin D and latrunculin B inhibited force-induced increases of SMA promoter activity. Deletion analyses showed that SMA promoter activity was increased approximately 70% after force with a minimal construct containing 155 bp upstream of the translation start site. The force effect on the SMA promoter was abrogated in cells transfected with CArG-B box mutants. Gel mobility shift analyses of nuclear extracts showed strong binding to the CArG-B motif after force. We conclude that the CArG-B box is a force-responsive element in the SMA promoter.