Swim training induces distinct osseous gene expression patterns in anosteocytic and osteocytic teleost fish.

The traditional understanding of bone mechanosensation implicates osteocytes, canaliculi, and the lacunocanalicular network in biomechanical adaptation. However, recent findings challenge this notion, as shown in advanced teleost fish where anosteocytic bone lacking osteocytes are nevertheless responsive to mechanical load. To investigate specific molecular mechanisms involved in bone mechanoadaptation in osteocytic and anosteocytic fish bone, we conducted a 5-min single swim-training experiment with zebrafish and ricefish, respectively. Through RNASeq analysis of fish spines, analyzed at various time points following swim training, we uncovered distinct gene expression patterns in osteocytic and anosteocytic fish bones. Notably, osteocytic fish bone exhibited an early response to mechanical load, contrasting to a delayed response observed in anosteocytic fish bones, both within 8 h following stimulation. We identified an increase in osteoblast differentiation in anosteocytic bone following training, while chordoblast activity was delayed. This temporal response suggests a time-dependent adaptation in anosteocytic bone, indicating the presence of intricate feedback networks within bone that lacks osteocytes.

Developmental trajectories of speech and language in neglected children aged 3 to 5 years: Results of the ELLAN study.

BACKGROUND: Neglected children are at high risk for significant difficulties in speech and language development. Because no longitudinal study has been conducted to date, the dynamic description of development during the preschool period is unknown. OBJECTIVES: Establish the developmental trajectories of speech sounds, receptive and expressive vocabulary, and morphosyntax among neglected children during the preschool years and compare them with those of non-neglected children. PARTICIPANTS AND SETTING: Participants are 69 neglected children and 99 same age non-neglected peers (37 and 46 males respectively) recruited at 36 months of age. Data were collected at home. METHODS: Data were collected at six-month intervals between the ages of 3 and 5.5 years using psychometrically robust tools. Neglected and control groups were compared according to age using repeated measures ANOVAs on all variables. A discrete mixture model for clustering longitudinal data was used for testing the heterogeneity of the language trajectories among neglected children. RESULTS: The language development of the neglected children as a whole group is lower than that of the control group for all variables. Two subgroups are identified within the neglected group: one with a developmental trajectory similar to that of the non-neglected children, and another whose trajectory is far below that of the control group. The effect sizes of these differences vary between 1.4 and 3 standard deviations under the mean. CONCLUSIONS: A large proportion of neglected children present significant speech and language difficulties from the age of 3, but some of them catch up and develop similarly to non-neglected children.

Breed and loading history influence in vivo skeletal strain patterns in pre-pubertal female chickens.

The influence of loading history on in vivo strains within a given specie remains poorly understood, and although in vivo strains have been measured at the hindlimb bones of various species, strains engendered during modes of activity other than locomotion are lacking, particularly in non-human species. For commercial egg-laying chickens specifically, there is an interest in understanding their bones' mechanical behaviour, particularly during youth, to develop early interventions to prevent the high incidence of osteoporosis in this population. We measured in vivo mechanical strains at the tibiotarsus midshaft during steady activities (ground, uphill, downhill locomotion) and non-steady activities (perching, jumping, aerial transition landing) in 48 pre-pubescent female (egg-laying) chickens from two breeds that were reared in three different housing systems, allowing varying amounts and types of physical activity. Mechanical strain patterns differed between breeds, and were dependent on the activity performed. Mechanical strains were also affected by rearing environment: chickens that were restricted from performing dynamic load bearing activity due to caged-housing generally exhibited higher mechanical strain levels during steady, but not non-steady activities, compared to chickens with prior dynamic load-bearing activity experience. Among chickens with prior experience of dynamic load bearing activity, those reared in housing systems that allowed more frequent physical activity did not exhibit lower mechanical strains. In all groups, the tibiotarsus was subjected to a loading environment consisting of a combination of axial compression, bending, and torsion, with torsion being the predominant source of strain. Aerial transition landing produced the highest strain levels with unusual strain patterns compared to other activities, suggesting it may produce the strongest anabolic response. These results exemplify how different breeds within a given specie adapt to maintain different patterns of mechanical strains, and how benefits of physical activity in terms of resistance to strain are activity-type dependent and do not necessarily increase with increased physical activity. These findings directly inform controlled loading experiments aimed at studying the bone mechanoresponse in young female chickens and can also be associated to measures of bone morphology and material properties to understand how these features influence bone mechanical properties in vivo.

3D Image Registration Marginally Improves the Precision of HR-pQCT Measurements Compared to Cross-Sectional-Area Registration in Adults With Osteogenesis Imperfecta.

Repositioning error in longitudinal high-resolution peripheral-quantitative computed tomography (HR-pQCT) imaging can lead to different bone volumes being assessed over time. To identify the same bone volumes at each time point, image registration is used. While cross-sectional area image registration corrects axial misalignment, 3D registration additionally corrects rotations. Other registration methods involving matched angle analysis (MA) or boundary transformations (3D-TB) can be used to limit interpolation error in 3D-registering micro-finite-element data. We investigated the effect of different image registration methods on short-term in vivo precision in adults with osteogenesis imperfecta, a collagen-related genetic disorder resulting in low bone mass, impaired quality, and increased fragility. The radii and tibiae of 29 participants were imaged twice on the same day with full repositioning. We compared the precision error of different image registration methods for density, microstructural, and micro-finite-element outcomes with data stratified based on anatomical site, motion status, and scanner generation. Regardless of the stratification, we found that image registration improved precision for total and trabecular bone mineral densities, trabecular and cortical bone mineral contents, area measurements, trabecular bone volume fraction, separation, and heterogeneity, as well as cortical thickness and perimeter. 3D registration marginally outperformed cross-sectional area registration for some outcomes, such as trabecular bone volume fraction and separation. Similarly, precision of micro-finite-element outcomes was improved after image registration, with 3D-TB and MA methods providing greatest improvements. Our regression model confirmed the beneficial effect of image registration on HR-pQCT precision errors, whereas motion had a detrimental effect on precision even after image registration. Collectively, our results indicate that 3D registration is recommended for longitudinal HR-pQCT imaging in adults with osteogenesis imperfecta. Since our precision errors are similar to those of healthy adults, these results can likely be extended to other populations, although future studies are needed to confirm this. © 2022 American Society for Bone and Mineral Research (ASBMR).

Bone adaptation to mechanical loading in mice is affected by circadian rhythms.

Physical forces are critical for successful function of many organs including bone. Interestingly, the timing of exercise during the day alters physiology and gene expression in many organs due to circadian rhythms. Circadian clocks in tissues, such as bone, express circadian clock genes that target tissue-specific genes, resulting in tissue-specific rhythmic gene expression (clock-controlled genes). We hypothesized that the adaptive response of bone to mechanical loading is regulated by circadian rhythms. First, mice were sham loaded and sacrificed 8 h later, which amounted to tissues being collected at zeitgeber time (ZT)2, 6, 10, 14, 18, and 22. Cortical bone of the tibiae collected from these mice displayed diurnal expression of core clock genes and key osteocyte and osteoblast-related genes, such as the Wnt-signaling inhibitors Sost and Dkk1, indicating these are clock-controlled genes. Serum bone turnover markers did not display rhythmicity. Second, mice underwent a single bout of in vivo loading at either ZT2 or ZT14 and were sacrificed 1, 8, or 24 h after loading. Loading at ZT2 resulted in Sost upregulation, while loading at ZT14 led to Sost and Dkk1 downregulation. Third, mice underwent daily in vivo tibial loading over 2 weeks administered either in the morning, (ZT2, resting phase) or evening (ZT14, active phase). In vivo microCT was performed at days 0, 5, 10, and 15 and conventional histomorphometry was performed at day 15. All outcome measures indicated a robust response to loading, but only microCT-based time-lapse morphometry showed that loading at ZT14 resulted in a greater endocortical bone formation response compared to mice loaded at ZT2. The decreased Sost and Dkk1 expression coincident with the modest, but significant time-of-day specific increase in adaptive bone formation, suggests that circadian clocks influence bone mechanoresponse.

Multisite longitudinal calibration of HR-pQCT scanners and precision in osteogenesis imperfecta.

BACKGROUND: For high-resolution peripheral quantitative computed tomography (HR-pQCT) to be used in longitudinal multi-center studies to assess disease and treatment effects, data must be aggregated across multiple timepoints and scanners. This requires an understanding of the factors contributing to scanner precision, and multi-scanner cross-calibration procedures, especially for clinical populations with severe phenotypes, like osteogenesis imperfecta (OI). METHODS: To address this, we first evaluated single- and multi-center short- and long-term precision errors of standard HR-pQCT parameters. Two imaging phantoms were circulated among 13 sites (7 XtremeCT and 6 XtremeCT2) and scanned in triplicate at 3 timepoints/site. Additionally, duplicate in vivo radial and tibial scans were acquired in 29 individuals with OI. Secondly, we investigated subject- and scanner-related factors that contribute to precision errors using regression analysis. Thirdly, we proposed a reference site selection criterion for multisite cross-calibration and demonstrated the external validity of phantom-based calibrations. RESULTS: Our results show excellent short-term single-site precision in both phantoms (CV % < 0.5%) and in density, microarchitecture and finite element parameters of OI participants (CV % = 0.75 to 1.2%). In vivo reproducibility significantly improved with (i) cross sectional area image registration versus no registration and (ii) scans with no motion artifacts. While reproducibility was similar across OI subtypes and anatomical sites, XtremeCT2 scanners achieved ~2.5% better precision than XtremeCT for trabecular parameters. Finally, we demonstrate that multisite longitudinal precision errors resulting from inconsistencies between scanners can be partially corrected through scanner cross-calibration. CONCLUSIONS: This study is the first to assess long-term reproducibility and cross-calibration in a study using first and second generation HR-pQCT scanners. The results presented in this context provide timely guidelines for future use of this powerful clinical imaging modality in multi-center longitudinal clinical trials.

Cortical bone adaptation to a moderate level of mechanical loading in male Sost deficient mice.

Loss-of-function mutations in the Sost gene lead to high bone mass phenotypes. Pharmacological inhibition of Sost/sclerostin provides a new drug strategy for treating osteoporosis. Questions remain as to how physical activity may affect bone mass under sclerostin inhibition and if that effect differs between males and females. We previously observed in female Sost knockout (KO) mice an enhanced cortical bone formation response to a moderate level of applied loading (900 µÎµ at the tibial midshaft). The purpose of the present study was to examine cortical bone adaptation to the same strain level applied to male Sost KO mice. Strain-matched in vivo compressive loading was applied to the tibiae of 10-, 26- and 52-week-old male Sost KO and littermate control (LC) mice. The effect of tibial loading on bone (re)modeling was measured by microCT, 3D time-lapse in vivo morphometry, 2D histomorphometry and gene expression analyses. As expected, Sost deficiency led to high cortical bone mass in 10- and 26-week-old male mice as a result of increased bone formation. However, the enhanced bone formation associated with Sost deficiency did not appear to diminish with skeletal maturation. An increase in bone resorption was observed with skeletal maturation in male LC and Sost KO mice. Two weeks of in vivo loading (900 µÎµ at the tibial midshaft) induced only a mild anabolic response in 10- and 26-week-old male mice, independent of Sost deficiency. A decrease in the Wnt inhibitor Dkk1 expression was observed 3 h after loading in 52-week-old Sost KO and LC mice, and an increase in Lef1 expression was observed 8 h after loading in 10-week-old Sost KO mice. The current results suggest that long-term inhibition of sclerostin in male mice does not influence the adaptive response of cortical bone to moderate levels of loading. In contrast with our previous strain-matched study in females showing enhanced bone responses with Sost ablation, these results in males indicate that the influence of Sost deficiency on the cortical bone formation response to a moderate level of loading differs between males and females. Clinical studies examining antibodies to inhibit sclerostin may need to consider that the efficacy of additional physical activity regimens may be sex dependent.

The half-life of the bone-derived hormone osteocalcin is regulated through O-glycosylation in mice, but not in humans.

Osteocalcin (OCN) is an osteoblast-derived hormone with pleiotropic physiological functions. Like many peptide hormones, OCN is subjected to post-translational modifications (PTMs) which control its activity. Here, we uncover O-glycosylation as a novel PTM present on mouse OCN and occurring on a single serine (S8) independently of its carboxylation and endoproteolysis, two other PTMs regulating this hormone. We also show that O-glycosylation increases OCN half-life in plasma ex vivo and in the circulation in vivo. Remarkably, in human OCN (hOCN), the residue corresponding to S8 is a tyrosine (Y12), which is not O-glycosylated. Yet, the Y12S mutation is sufficient to O-glycosylate hOCN and to increase its half-life in plasma compared to wildtype hOCN. These findings reveal an important species difference in OCN regulation, which may explain why serum concentrations of OCN are higher in mouse than in human.

Bones provide support and protection for organs in the body. However, over the last 15 years researchers have discovered that bones also release chemicals known as hormones, which can travel to other parts of the body and cause an effect. The cells responsible for making bone, known as osteoblasts, produce a hormone called osteocalcin which communicates with a number of different organs, including the pancreas and brain. When osteocalcin reaches the pancreas, it promotes the release of another hormone called insulin which helps regulate the levels of sugar in the blood. Osteocalcin also travels to other organs such as muscle, where it helps to degrade fats and sugars that can be converted into energy. It also has beneficial effects on the brain, and has been shown to aid memory and reduce depression. Osteocalcin has largely been studied in mice where levels are five to ten times higher than in humans. But it is unclear why this difference exists or how it alters the role of osteocalcin in humans. To answer this question, Al Rifai et al. used a range of experimental techniques to compare the structure and activity of osteocalcin in mice and humans. The experiments showed that mouse osteocalcin has a group of sugars attached to its protein structure, which prevent the hormone from being degraded by an enzyme in the blood. Human osteocalcin has a slightly different protein sequence and is therefore unable to bind to this sugar group. As a result, the osteocalcin molecules in humans are less stable and cannot last as long in the blood. Al Rifai et al. showed that when human osteocalcin was modified so the sugar group could attach, the hormone was able to stick around for much longer and reach higher levels when added to blood in the laboratory. These findings show how osteocalcin differs between human and mice. Understanding this difference is important as the effects of osteocalcin mean this hormone can be used to treat diabetes and brain disorders. Furthermore, the results reveal how the stability of osteocalcin could be improved in humans, which could potentially enhance its therapeutic effect.

Morphosyntactic Development and Severe Parental Neglect in 4-Year-Old French-Speaking Children: ELLAN study.

Language is the most frequently compromised area of development in English-speaking neglected children, particularly the morphosyntactic component of language. This is very worrisome given its central role in academic success and social participation. No previous study has examined the morphosyntactic skills of French-speaking neglected children, despite the morphological richness of French. This study aimed to fill this gap. Forty-four neglected (mean age = 48.32 months, SD = 0.45) and 92 non-neglected (mean age = 48.07 months, SD = 0.24) French-speaking children participated. Measures of morphosyntactic skills were derived from a sample of spontaneous language collected during standardized semistructured play and analyzed using Systematic Analysis of Language Transcripts software (2012) . Four morphosyntactic indicators were compared using analyses of variance and Kolmogorov-Smirnov tests: the mean length of utterances (MLU), verbal inflections, word-level errors, and omission errors. The results indicate that 25.6% of the neglected children presented clinically significant morphosyntactic difficulties, as evidenced by a significantly shorter MLU (M = 5.60, SD = 1.13; M = 6.90, SD = 1.30), fewer verbal inflections, and more frequent word omission errors compared to their non-neglected peers. The results confirm that French-speaking neglected children present many morphosyntactic difficulties. This study argues for sustained speech-language services for these children.

Sclerostin Neutralizing Antibody Treatment Enhances Bone Formation but Does Not Rescue Mechanically Induced Delayed Healing.

During bone healing, tissue formation processes are governed by mechanical strain. Sost/sclerostin, a key Wnt signaling inhibitor and mechano-sensitive pathway, is downregulated in response to mechanical loading. Sclerostin neutralizing antibody (SclAb) increases bone formation. Nevertheless, it remains unclear whether sclerostin inhibition can rescue bone healing in situations of mechanical instability, which otherwise delay healing. We investigated SclAb's influence on tissue formation in a mouse femoral osteotomy, stabilized with rigid or semirigid external fixation. The different fixations allowed different magnitudes of interfragmentary movement during weight bearing, thereby influencing healing outcome. SclAb or vehicle (veh) was administeredand bone healing was assessed at multiple time points up to day 21 postoperatively by in vivo micro-computed tomography, histomorphometry, biomechanical testing, immunohistochemistry, and gene expression. Our results show that SclAb treatment caused a greater bone volume than veh. However, SclAb could not overcome the characteristic delayed healing of semirigid fixation. Indeed, semirigid fixation resulted in delayed healing with a prolonged endochondral ossification phase characterized by increased cartilage, lower bone volume fraction, and less bony bridging across the osteotomy gap than rigid fixation. In a control setting, SclAb negatively affected later stages of healing under rigid fixation, evidenced by the high degree of endosteal bridging at 21 days in the rigid-SclAb group compared with rigid-veh, indicating delayed fracture callus remodeling and bone marrow reconstitution. Under rigid fixation, Sost and sclerostin expression at the gene and protein level, respectively, were increased in SclAb compared with veh-treated bones, suggesting a negative feedback mechanism. Our results suggest that SclAb could be used to enhance overall bone mass but should be carefully considered in bone healing. SclAb may help to increase bone formation early in the healing process but not during advanced stages of fracture callus remodeling and not to overcome delayed healing in semirigid fixation. © 2018 American Society for Bone and Mineral Research.

Proprotein convertase furin regulates osteocalcin and bone endocrine function.

Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its γ-carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function.

Sost deficiency led to a greater cortical bone formation response to mechanical loading and altered gene expression.

Bone adaptation optimizes mass and structure, but the mechano-response is already reduced at maturation. Downregulation of sclerostin was believed to be a mandatory step in mechano-adaptation, but in young mice it was shown that load-induced formation can occur independent of sclerostin, a product of the Sost gene. We hypothesized that the bone formation and resorption response to loading is not affected by Sost deficiency, but is age-specific. Our findings indicate that the anabolic response to in vivo tibial loading was reduced at maturation in Sost Knockout (KO) and littermate control (LC) mice. Age affected all anabolic and catabolic parameters and altered Sost and Wnt target gene expression. While load-induced cortical resorption was similar between genotypes, loading-induced gains in mineralizing surface was enhanced in Sost KO compared to LC mice. Loading led to a downregulation in expression of the Wnt inhibitor Dkk1. Expression of Dkk1 was greater in both control and loaded limbs of Sost KO compared to LC mice suggesting a compensatory role in the absence of Sost. These data suggest physical activity could enhance bone mass concurrently with sclerostin-neutralizing antibodies, but treatment strategies should consider the influence of age on ultimate load-induced bone mass gains.

Genetic demonstration of a redundant role of extracellular signal-regulated kinase 1 (ERK1) and ERK2 mitogen-activated protein kinases in promoting fibroblast proliferation.

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway plays an important role in the proliferative response of mammalian cells to mitogens. However, the individual contribution of the isoforms ERK1 and ERK2 to cell proliferation control is unclear. The two ERK isoforms have similar biochemical properties and recognize the same primary sequence determinants on substrates. On the other hand, analysis of mice lacking individual ERK genes suggests that ERK1 and ERK2 may have evolved unique functions. In this study, we used a robust genetic approach to analyze the individual functions of ERK1 and ERK2 in cell proliferation using genetically matched primary embryonic fibroblasts. We show that individual loss of either ERK1 or ERK2 slows down the proliferation rate of fibroblasts to an extent reflecting the expression level of the kinase. Moreover, RNA interference-mediated silencing of ERK1 or ERK2 expression in cells genetically disrupted for the other isoform similarly reduces cell proliferation. We generated fibroblasts genetically deficient in both Erk1 and Erk2. Combined loss of ERK1 and ERK2 resulted in a complete arrest of cell proliferation associated with G(1) arrest and premature replicative senescence. Together, our findings provide compelling genetic evidence for a redundant role of ERK1 and ERK2 in promoting cell proliferation.

Phosphorylation of Ser72 does not regulate the ubiquitin ligase activity and subcellular localization of Skp2.

Skp2 is the substrate binding subunit of the SCF(Skp2) ubiquitin ligase, which plays a key role in the regulation of cell cycle progression. The activity of Skp2 is regulated by the APC(Cdh1), which targets Skp2 for degradation in early G(1) and prevent premature S phase entry. Overexpression of Skp2 leads to dysregulation of the cell cycle and is commonly observed in human cancers. We have previously shown that Skp2 is phosphorylated on Ser64 and Ser72 in vivo, and that these modifications regulate its stability. Recently, two studies have proposed a role for Ser72 phosphorylation in the cytosolic relocalization of Skp2 and in the assembly and activity of SCF(Skp2) ubiquitin ligase complex. We have revisited this question and analyzed the impact of Ser72 phosphorylation site mutations on the biological activity and subcellular localization of Skp2. We show here that phosphorylation of Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2 in a panel of cell lines.

Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors.

BACKGROUND: The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established. METHODS: Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells. RESULTS: We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect. CONCLUSION: MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.

Regulation of MAPK-activated protein kinase 5 activity and subcellular localization by the atypical MAPK ERK4/MAPK4.

MAPK-activated protein kinase 5 (MK5) was recently identified as a physiological substrate of the atypical MAPK ERK3. Complex formation between ERK3 and MK5 results in phosphorylation and activation of MK5, concomitant stabilization of ERK3, and the nuclear exclusion of both proteins. However, ablation of ERK3 in HeLa cells using small interfering RNA or in fibroblasts derived from ERK3 null mice reduces the activity of endogenous MK5 by only 50%, suggesting additional mechanisms of MK5 regulation. Here we identify the ERK3-related kinase ERK4 as a bona fide interaction partner of MK5. Binding of ERK4 to MK5 is accompanied by phosphorylation and activation of MK5. Furthermore, complex formation also results in the relocalization of MK5 from nucleus to cytoplasm. However unlike ERK3, ERK4 is a stable protein, and its half-life is not modified by the presence or absence of MK5. Finally, although knock-down of ERK4 protein in HeLa cells reduces endogenous MK5 activity by approximately 50%, a combination of small interfering RNAs targeting both ERK4 and ERK3 causes a further reduction in the MK5 activity by more than 80%. We conclude that MK5 activation is dependent on both ERK3 and ERK4 in these cells and that these atypical MAPKs are both physiological regulators of MK5 activity.

Cyclic AMP induces morphological changes of vascular smooth muscle cells by inhibiting a Rac-dependent signaling pathway.

Cyclic AMP (cAMP) is a pleiotropic second messenger that regulates numerous cellular processes. In vascular smooth muscle cells (VSMCs), these include cell proliferation, migration, and contractility. Here we show that cAMP-elevating agents induce dramatic morphological changes in VSMCs, characterized by cell rounding and formation of long branching processes. The stellate morphology is associated with disassembly of actin stress fibers and lamellipodia, loss of focal adhesions, and the formation of small F-actin rings. Because of the importance of Rho family GTPases in regulating actin dynamics, we analyzed their individual roles in the cAMP phenotype. We found that pharmacological or genetic inhibition of Rac mimics cAMP effect in inducing a stellate morphology of VSMCs. Expression of activated Rac1 prevents forskolin-induced cAMP stellation, suggesting that cAMP affects cell morphology by inhibiting Rac function. Consistent with this, treatment with forskolin inhibits agonist-stimulated Rac activation in VSMCs. We further show that activated Rac1 containing the F37A effector loop substitution fails to rescue the cAMP phenotype. Our results suggest that cAMP modulates the morphology of VSMCs by inhibiting a Rac-dependent signaling pathway.

Nuclear export of ERK3 by a CRM1-dependent mechanism regulates its inhibitory action on cell cycle progression.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase family of serine/threonine kinases. Little is known on the regulation of ERK3 function. Here, we report that ERK3 is constitutively localized in the cytoplasmic and nuclear compartments. In contrast to other mitogen-activated protein kinases, the cellular distribution of ERK3 remains unchanged in response to common mitogenic or stress stimuli and is independent of the enzymatic activity or phosphorylation of the kinase. The cytoplasmic localization of ERK3 is directed by a CRM1-dependent nuclear export mechanism. Treatment of cells with leptomycin B causes the nuclear accumulation of ERK3 in a high percentage of cells. Moreover, ectopic expression of CRM1 promotes the cytoplasmic relocalization of ERK3, whereas overexpression of snurportin 1, which binds CRM1 with high affinity, inhibits the nuclear export of ERK3. We also show that CRM1 binds to ERK3 in vitro. Importantly, we show that enforced localization of ERK3 in the nucleus or cytoplasm markedly attenuates the ability of the kinase to induce cell cycle arrest in fibroblasts. Our results suggest that nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression.