Amniotic Fluid-derived MSC Secretome Halts Action of IL-1β and TNF-α Through ERK/MAPK and Returns Cartilage Repair Under OA Inflammatory Stimuli

Background: Osteoarthritis (OA) is a degenerative cartilage disease. OA cartilage has a limited repair capacity due to the effect of IL-1β and TNF-α on the chondrocyte progenitor cells (CPC) in an OA joint. Mesenchymal stem cells (MSC) therapy is a therapeutic option for osteoarthritis that initiated by the ability of secretory growth factors and mediator molecules to heal OA. Amniotic fluid MSC (AF-MSC), an interesting MSC source, has been shown to secrete various growth factors and anti-inflammatory molecules promoting tissue repair and regeneration. However, the effect of AF-MSC secretory factors to inflammation and cartilage repair is still limited. The current study aims to explore the action of AF-MSC secretome to IL-1β and TNF-α, and the CPC function that encourages cartilage repair.Methods: The effect of AF-MSC secretome to OA inflammatory cytokines was observed via the CPC migration using scratch assay. Inhibitory action of AF-MSC secretome to IL-1β and TNF-α was determined through NF-κB and MAPK signaling pathways by western blot. The repaired function of OA cartilage was analyzed via the cartilage outgrowth study and the expression of chondrogenic and anabolic genes using qRT-PCR.Results: AF-MSC secretome can arrest inflammatory action of IL-1β and TNF-α and reduces production of NF-κB, pNF-κB, p38, pp38, ERK, COX-2, and iNOS signaling proteins. It significantly reduced the production of pERK (P = 0.0434). For cartilage repair, AF-MSC secretome promotes CPC outgrowth and migration in human OA cartilage, even under inflammatory stimuli. By the action of AF-MSC secretome, the inflamed CPC can restore Col II and anabolic genes; IGF1 expression, indicating reactivation of cartilage regeneration.Conclusion: AF-MSC secretory factors have the ability to halt inflammatory actions of IL-1β and TNF-α via the ERK/MAPK pathway and motivate CPC function and anabolic property.

OPN Inhibits Autophagy through CD44, Integrin and the MAPK Pathway in Osteoarthritic Chondrocytes

Background: To investigate whether OPN has an effect on autophagy in human osteoarthritic chondrocytes and determine the roles of CD44, αvβ3 integrin and the MAPK pathway in this progress. Methods: First, we cultured human OA chondrocytes in vitro and then treated cells with rhOPN to determine autophagy changes. Next , the anti-CD44 and anti-CD51/61 monoclonal antibodies (Abs) or isotype IgG were used to determine the possible role of CD44 and αvβ3 integrin; subsequently, an inhibitor of the ERK MAPK pathway was used to investigate the role of ERK MAPK. Western blotting was used to measure the beclin1, LC3 II and MAPK protein expression, and mRFP-GFP-LC3 confocal imaging was used to detect the autophagy levels. CCK-8 was used to assay the proliferation and activity of chondrocytes. Results: Our results showed that the LC3 protein was greatly decreased in OA cartilage compared to normal cartilage ,and OPN suppressed the autophagy activity in chondrocytes in vitro. Blocking experiments with anti-CD44 and anti-CD51/61 Abs indicated that OPN could suppress the expression of LC3II and beclin1 through αvβ3 integrin and CD44. Our results also indicated that the ratio of p-ERK/ ERK but not p-P38/P38 and p-JNK/JNK was increased after the rhOPN treatment. The ERK inhibitor inhibited the activity of OPN in the suppression of autophagy, and the CCK-8 results showed that rhOPN could promote chondrocyte proliferation. Conclusions: OPN inhibited chondrocyte autophagy through CD44 and αvβ3 integrin receptors and via the ERK MAPK signaling pathway.

Shexiang Tongxin Dropping Pill Protects Against Chronic Heart Failure in Mice via Inhibiting the ERK/MAPK and TGF-β Signaling Pathways

Background: Chronic heart failure (CHF) is a major public health problem with high mortality and morbidity worldwide. Shexiang Tongxin Dropping Pill (STDP) is a widely used traditional Chinese medicine preparation for coronary heart disease and growing evidence proves that STDP exerts beneficial effects on CHF in the clinic. However, the molecular mechanism of the therapeutic effects of STDP on CHF remains largely unknown.Objective: This study aimed to elucidate the mechanism of action of STDP against CHF by integrating network pharmacology analysis and whole-transcriptome sequencing.Methods: First, the mouse model of CHF was established by the transverse aortic constriction (TAC) surgery, and the efficacy of STDP against CHF was evaluated by assessing the alterations in cardiac function, myocardial fibrosis, and cardiomyocyte hypertrophy with echocardiography, Masson’s trichrome staining, and wheat germ agglutinin staining. Next, a CHF disease network was constructed by integrating cardiovascular disease-related genes and the transcriptome sequencing data, which was used to explore the underlying mechanism of action of STDP. Then, the key targets involved in the effects of STDP on CHF were determined by network analysis algorithms, and pathway enrichment analysis was performed to these key genes. Finally, important targets in critical pathway were verified in vivo.Results: STDP administration obviously improved cardiac function, relieved cardiomyocyte hypertrophy, and ameliorated myocardial fibrosis in CHF mice. Moreover, STDP significantly reversed the imbalanced genes that belong to the disease network of CHF in mice with TAC, and the number of genes with the reverse effect was 395. Pathway analysis of the crucial genes with recovery efficiency revealed that pathways related to fibrosis and energy metabolism were highly enriched, while TGF-β pathway and ERK/MAPK pathway were predicted to be significantly affected. Consistently, validation experiments confirmed that inhibiting ERK/MAPK and TGF-β signaling pathways via reduction of the phosphorylation level of Smad3 and ERK1/2 is the important mechanism of STDP against CHF.Conclusion: Our data demonstrated that STDP can recover the imbalanced CHF network disturbed by the modeling of TAC through the multi-target and multi-pathway manner in mice, and the mechanisms are mainly related to inhibition of ERK/MAPK and TGF-β signaling pathways.

A role for the Erk MAPK pathway in modulating SAX-7/L1CAM-dependent locomotion in Caenorhabditis elegans

L1CAMs are immunoglobulin cell adhesion molecules that function in nervous system development and function. Besides being associated with autism and schizophrenia spectrum disorders, impaired L1CAM function also underlies the X-linked L1 syndrome, which encompasses a group of neurological conditions, including spastic paraplegia and congenital hydrocephalus. Studies on vertebrate and invertebrate L1CAMs established conserved roles that include axon guidance, dendrite morphogenesis, synapse development, and maintenance of neural architecture. We previously identified a genetic interaction between the C. elegans L1CAM encoded by the sax-7 gene and RAB-3, a GTPase that functions in synaptic neurotransmission; rab-3; sax-7 mutant animals exhibit synthetic locomotion abnormalities and neuronal dysfunction. Here, we show that this synergism also occurs when loss of SAX-7 is combined with mutants of other genes encoding key players of the synaptic vesicle cycle. In contrast, sax-7 does not interact with genes that function in synaptogenesis. These findings suggest a post-developmental role for sax-7 in the regulation of synaptic activity. To assess this possibility, we conducted electrophysiological recordings and ultrastructural analyses at neuromuscular junctions; these analyses did not reveal obvious synaptic abnormalities. Lastly, based on a forward genetic screen for suppressors of the rab-3; sax-7 synthetic phenotypes, we determined that mutants in the ERK Mitogen-activated Protein Kinase (MAPK) pathway can suppress the rab-3; sax-7 locomotion defects. Moreover, we established that Erk signaling acts in a subset of cholinergic neurons in the head to promote coordinated locomotion. In combination, these results suggest a modulatory role for Erk MAPK in L1CAM-dependent locomotion in C. elegans.

Expression of Spred2 in the urothelial tumorigenesis of the urinary bladder

Aberrant activation of the Ras/Raf/ERK (extracellular-signal-regulated kinase)-MAPK (mitogen-activated protein kinase) pathway is involved in the progression of cancer, including urothelial carcinoma; but the negative regulation remains unclear. In the present study, we investigated pathological expression of Spred2 (Sprouty-related EVH1 domain-containing protein 2), a negative regulator of the Ras/Raf/ERK-MAPK pathway, and the relation to ERK activation and Ki67 index in various categories of 275 urothelial tumors obtained from clinical patients. In situ hybridization demonstrated that Spred2 mRNA was highly expressed in high-grade non-invasive papillary urothelial carcinoma (HGPUC), and the expression was decreased in carcinoma in situ (CIS) and infiltrating urothelial carcinoma (IUC). Immunohistochemically, membranous Spred2 expression, important to interact with Ras/Raf, was preferentially found in HGPUC. Interestingly, membranous Spred2 expression was decreased in CIS and IUC relative to HGPUC, while ERK activation and the expression of the cell proliferation marker Ki67 index were increased. HGPUC with membranous Spred2 expression correlated significantly with lower levels of ERK activation and Ki67 index as compared to those with negative Spred2 expression. Thus, our pathological findings suggest that Spred2 counters cancer progression in non-invasive papillary carcinoma possibly through inhibiting the Ras/Raf/ERK-MAPK pathway, but this regulatory mechanism is lost in cancers with high malignancy. Spred2 appears to be a key regulator in the progression of non-invasive bladder carcinoma.

Smad Interacting Protein-1 is Essential for Oligodendrocyte Differentiation

Precursor/stem cell substitutive therapy to promote remyelination is an ideal strategy for central nervous system demyelinating diseases such as spinal cord injury (SCI). However, the microenvironment of the injured area is not conducive to the survival, differentiation, and functions of the transplanted cells. Identifying and regulating the key inhibitory factors might be an important target for the treatment of demyelinating diseases. Smad interacting protein-1 (Sip1) is a transcription factor that binds to phosphorylated R-Smad in the nucleus, which promotes remyelination by inducing the differentiation of oligodendrocytes. In this study, we show that the expression of Sip1 is up-regulated and peaks by 1 day and then returns to normal levels 7 days after SCI. Most Sip1 positive cells were oligodendrocytes. In vitro, Sip1 was weakly expressed in the cytoplasm of oligodendrocyte progenitor cells (OPCs), significantly up-regulated in immature oligodendrocytes, and showed significant nuclear transposition. In contrast, Sip1 expression levels in mature oligodendrocytes decreased to levels similar to those in OPCs. The RNA interference of Sip1 in OPCs reduced the level of myelin basic protein (a mature oligodendrocyte marker protein, MBP) and pERK1/2 (a key molecule of the ERK/MAPK pathway) in oligodendrocytes. These findings suggest that Sip1 is essential for oligodendrocyte differentiation and might affect the ERK/MAPK signal pathway. The results provide a theoretical basis for the treatment of demyelinating lesions such as spinal cord injury by regulating Sip1 expression in oligodendrocytes.

Dynamic m6A mRNA Methylation Reveals the Role of METTL3/14-m6A-MNK2-ERK Signaling Axis in Skeletal Muscle Differentiation and Regeneration

N6-methyladenosine (m6A) RNA methylation has emerged as an important factor in various biological processes by regulating gene expression. However, the dynamic profile, function and underlying molecular mechanism of m6A modification during skeletal myogenesis remain elusive. Here, we report that members of the m6A core methyltransferase complex, METTL3 and METTL14, are downregulated during skeletal muscle development. Overexpression of either METTL3 or METTL14 dramatically blocks myotubes formation. Correspondingly, knockdown of METTL3 or METTL14 accelerates the differentiation of skeletal muscle cells. Genome-wide transcriptome analysis suggests ERK/MAPK is the downstream signaling pathway that is regulated to the greatest extent by METTL3/METTL14. Indeed, METTL3/METTL14 expression facilitates ERK/MAPK signaling. Via MeRIP-seq, we found that MNK2, a critical regulator of ERK/MAPK signaling, is m6A modified and is a direct target of METTL3/METTL14. We further revealed that YTHDF1 is a potential reader of m6A on MNK2, regulating MNK2 protein levels without affecting mRNA levels. Furthermore, we discovered that METTL3/14-MNK2 axis was up-regulated notably after acute skeletal muscle injury. Collectively, our studies revealed that the m6A writers METTL3/METTL14 and the m6A reader YTHDF1 orchestrate MNK2 expression posttranscriptionally and thus control ERK signaling, which is required for the maintenance of muscle myogenesis and may contribute to regeneration.