ERK3‐MK5 Signaling in Murine Ventricular Fibroblasts and its Role in Fibroblast Migration
Bibliographic record
Abstract
Background and objectives MAP kinase‐activated protein kinase‐5 (MK5), a protein serine/threonine kinase identified as a substrate for p38α/β, ERK3, and ERK4 MAPKs, is expressed in the heart. However, the interacting partners and physiological function of MK5 are just beginning to be understood. Both MK5 and ERK3 haploinsufficiency attenuates the increase in cardiac collagen 1‐α 1 mRNA induced by constriction of the transverse aorta. Additionally, following myocardial ischemia induced by ligation of the left anterior descending coronary artery, scar size and collagen content were reduced in MK5 haploinsufficient mice compared with wildtype littermates. This study was to examine ERK3‐MK5 signaling in cardiac ventricular fibroblasts. Methods Cardiac fibroblasts were isolated from MK5 +/+ and MK5 −/− mice and male Sprague‐Dawley rats. Subconfluent cultures of fibroblasts from passages 2 and 3 were used. Protein expression, as well as the ratio of filamentous (F) to monomeric (G) actin, was determined by immunoblotting. The presence of ERK3‐MK5 complexes was determined by proximity ligation assay (PLA) and co‐immunoprecipitation assays. Cell motility and myofibroblast contraction were studied by scratch‐wound and collagen gel contraction assays, respectively. The subcellular distribution of ERK3, MK5, smooth muscle α‐actin (αSMA), and F‐actin was determined by immunocytofluorescence and confocal microscopy. Results MK5 immunoreactivity was primarily localized in the nucleus whereas that of ERK3 was in the cytoplasm. In actively dividing fibroblasts, phospho‐MK5 immunoreactivity appeared to be associated with the cytoskeleton and pseudopodia. Following serum stimulation, ERK3 immunoreactivity redistributed to membrane ruffles and/or lamellipodia. ERK3, but not ERK4 or p38α, immunoreactivity was detected in MK5 immunoprecipitates from fibroblast lysates. PLA revealed ERK3‐MK5 complexes in the cytoplasm, which were less abundant following siRNA‐mediated knockdown of MK5 (MK5‐kd). MK5‐kd cells showed reduced contraction of collagen gels, cell spreading, and formation of dendritic extensions. The ratio of F‐actin to G‐actin decreased upon MK5 knockdown. Furthermore. confocal fluorescence microscopy revealed a reduction in F‐actin in MK5‐kd fibroblasts compared to fibroblasts transfected with scrambled RNA. MK5 +/+ fibroblasts contained prominent, intersecting bundles of αSMA immunoreactivity in the leading edge of the cells, whereas MK5 −/− fibroblasts had thin, parallel bundles of αSMA in this region. Cell migration, in response to serum, and/or angiotensin II, was reduced upon siRNA‐mediated knockdown of either MK5 or ERK3. Conclusion Co‐immunoprecipitation and PLA suggest that ERK3 and MK5 form complexes in cardiac fibroblasts and that these complexes are located in the cytosol. Following serum stimulation, phospho‐MK5 and ERK3 immunoreactivity redistributed to the leading edges of the membranes. Suppressing ERK3 or MK5 expression decreased cell motility. Hence, MK5 and ERK3 signaling may play overlapping roles in regulating cardiac fibroblast function. Support or Funding Information This study was supported by a grant from the Heart and Stroke Foundation of Canada. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".