Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells

Background: The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK) play pivotal roles in cell migration, apoptosis (membrane blebbing), cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined. Methodology/Principal Findings: P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a \u27ground\u27 state after the inhibition had been removed. Conclusions/Significance: Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the role of the Rho-ROCK pathway in early cellular \u27fate\u27 decision making processes. © 2011 Krawetz et al

Inhibition of Rho Kinase Regulates Specification of Early Differentiation Events in P19 Embryonal Carcinoma Stem Cells

The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK) play pivotal roles in cell migration, apoptosis (membrane blebbing), cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined.P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a 'ground' state after the inhibition had been removed.Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the role of the Rho-ROCK pathway in early cellular 'fate' decision making processes

A Na\u3csup\u3e+\u3c/sup\u3e/K\u3csup\u3e+\u3c/sup\u3e ATPase Pump Regulates Chondrocyte Differentiation and Bone Length Variation in Mice

The genetic and developmental mechanisms involved in limb formation are relatively well documented, but how these mechanisms are modulated by changes in chondrocyte physiology to produce differences in limb bone length remains unclear. Here, we used high throughput RNA sequencing (RNAseq) to probe the developmental genetic basis of variation in limb bone length in Longshanks, a mouse model of experimental evolution. We find that increased tibia length in Longshanks is associated with altered expression of a few key endochondral ossification genes such as Npr3, Dlk1, Sox9, and Sfrp1, as well reduced expression of Fxyd2, a facultative subunit of the cell membrane-bound Na+/K+ ATPase pump (NKA). Next, using murine tibia and cell cultures, we show a dynamic role for NKA in chondrocyte differentiation and in bone length regulation. Specifically, we show that pharmacological inhibition of NKA disrupts chondrocyte differentiation, by upregulating expression of mesenchymal stem cell markers (Prrx1, Serpina3n), downregulation of chondrogenesis marker Sox9, and altered expression of extracellular matrix genes (e.g., collagens) associated with proliferative and hypertrophic chondrocytes. Together, Longshanks and in vitro data suggest a broader developmental and evolutionary role of NKA in regulating limb length diversity

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin\u27s ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology

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ABSTRACT. Objective. To determine whether cytokine/chemokine profiles from synovial fluid and sera discriminate mild/moderate osteoarthritis (OA) from normal and severe OA cohorts. Methods. Multiplex technology was used to quantify expression levels for 42 cytokines in the synovial fluid of patients diagnosed with severe OA (n = 20) and mild/moderate OA (n = 12), as well as normal controls (n = 34). The same 42 cytokines were examined in serum samples of patients with severe OA (n = 26) and mild/moderate OA (n = 74) and normal individuals (n = 100)

Resetting the clock on arthritis

Abstract While many of intricacies of the mammalian circadian rhythm remain unknown, the role that this physiological clock plays in our everyday lives and within the global ecosystem is clearly evident. However, only recently has the importance of circadian rhythm in cartilage health and joint homeostasis been brought to light. A recent study by Hand and colleagues advances our understanding of these processes further by demonstrating that disruption of circadian clock regulation in mesenchymal cells not only has an impact on the development of joint structures, but on the inflammatory response and on the onset/pathogenesis of arthritis

Understanding cartilage protection in OA and injury: a spectrum of possibilities

Abstract Background Osteoarthritis (OA) is a prevalent musculoskeletal disease resulting in progressive degeneration of the hyaline articular cartilage within synovial joints. Current repair treatments for OA often result in poor quality tissue that is functionally ineffective compared to the hyaline cartilage and demonstrates increased failure rates post-treatment. Complicating efforts to improve clinical outcomes, animal models used in pre-clinical research show significant heterogeneity in their regenerative and degenerative responses associated with their species, age, genetic/epigenetic traits, and context of cartilage injury or disease. These can lead to variable outcomes when testing and validating novel therapeutic approaches for OA. Furthermore, it remains unclear whether protection against OA among different model systems is driven by inhibition of cartilage degeneration, enhancement of cartilage regeneration, or any combination thereof. Main text Understanding the mechanistic basis underlying this context-dependent duality is essential for the rational design of targeted cartilage repair and OA therapies. Here, we discuss some of the critical variables related to the cross-species paradigm of degenerative and regenerative abilities found in pre-clinical animal models, to highlight that a gradient of regenerative competence within cartilage may exist across species and even in the greater human population, and likely influences clinical outcomes. Conclusions A more complete understanding of the endogenous regenerative potential of cartilage in a species specific context may facilitate the development of effective therapeutic approaches for cartilage injury and/or OA

Reduction of pluripotent gene expression in murine embryonic stem cells exposed to mechanical loading or Cyclo RGD peptide

Abstract Background Self-renewal and differentiation of embryonic stem cells (ESCs) is directed by biological and/or physical cues that regulate multiple signaling cascades. We have previously shown that mESCs seeded in a type I collagen matrix demonstrate a loss of pluripotent marker expression and differentiate towards an osteogenic lineage. In this study, we examined if this effect was mediated in part through Arginylglycylaspartic acid (RGD) dependent integrin activity and/or mechano-transduction. Results The results from this study suggest that mESC interaction with the local microenvironment through RGD dependent integrins play a role in the regulation of mESC core transcription factors (TF), Oct-4, Sox 2 and Nanog. Disruption of this interaction with a cyclic RGD peptide (cRGDfC) was sufficient to mimic the effect of a mechanical stimulus in terms of pluripotent gene expression, specifically, we observed that supplementation with cRGDfC, or mechanical stimulus, significantly influenced mESC pluripotency by down-regulating core transcription factors. Moreover, our results indicated that the presence of the cRGDfC peptide inhibited integrin expression and up-regulated early lineage markers (mesoderm and ectoderm) in a Leukemia inhibitory factor (LIF) dependent manner. When cRGDfC treated mESCs were injected in Severe combined immunodeficiency (SCID) mice, no tissue growth and/or teratoma formation was observed, suggesting that the process of mESC tumor formation in vivo is potentially dependent on integrin interaction. Conclusions Overall, the disruption of cell-integrin interaction via cRGDfC peptide can mimic the effect of mechanical stimulation on mESC pluripotency gene expression and also inhibit the tumorigenic potential of mESCs in vivo