Inhibition of c-Jun NH2-terminal kinase stimulates mu opioid receptor expression via p38 MAPK-mediated nuclear NF-κB activation in neuronal and non-neuronal cells

AbstractDespite its potential side effects of addiction, tolerance and withdrawal symptoms, morphine is widely used for reducing moderate and severe pain. Previous studies have shown that the analgesic effect of morphine depends on mu opioid receptor (MOR) expression levels, but the regulatory mechanism of MOR is not yet fully understood. Several in vivo and in vitro studies have shown that the c-Jun NH2-terminal kinase (JNK) pathway is closely associated with neuropathic hyperalgesia, which closely resembles the neuroplastic changes observed with morphine antinociceptive tolerance. In this study, we show that inhibition of JNK by SP600125, its inhibitory peptide, or JNK-1 siRNA induced MOR at both mRNA and protein levels in neuronal cells. This increase in MOR expression was reversed by inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway, but not by inhibition of the mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway. Further experiments using cell signaling inhibitors showed that MOR upregulation by JNK inhibition involved nuclear factor-kappa B (NF-κB). The p38 MAPK dependent phosphorylation of p65 NF-κB subunit in the nucleus was increased by SP600125 treatment. We also observed by chromatin immunoprecipitation (ChIP) analysis that JNK inhibition led to increased bindings of CBP and histone-3 dimethyl K4, and decreased bindings of HDAC-2, MeCP2, and histone-3 trimethyl K9 to the MOR promoter indicating a transcriptional regulation of MOR by JNK inhibition. All these results suggest a regulatory role of the p38 MAPK and NF-κB pathways in MOR gene expression and aid to our better understanding of the MOR gene regulation

Canonical Notch signaling is required for bone morphogenetic protein‐mediated human osteoblast differentiation

Osteoblast differentiation of bone marrow‐derived human mesenchymal stem cells (hMSC) can be induced by stimulation with canonical Notch ligand, Jagged1, or bone morphogenetic proteins (BMPs). However, it remains elusive how these two pathways lead to the same phenotypic outcome. Since Runx2 is regarded as a master regulator of osteoblastic differentiation, we targeted Runx2 with siRNA in hMSC. This abrogated both Jagged1 and BMP2 mediated osteoblastic differentiation, confirming the fundamental role for Runx2. However, while BMP stimulation increased Runx2 and downstream Osterix protein expression, Jagged1 treatment failed to upregulate either, suggesting that canonical Notch signals require basal Runx2 expression. To fully understand the transcriptomic profile of differentiating osteoblasts, RNA sequencing was performed in cells stimulated with BMP2 or Jagged1. There was common upregulation of ALPL and extracellular matrix genes, such as ACAN, HAS3, MCAM, and OLFML2B. Intriguingly, genes encoding components of Notch signaling (JAG1, HEY2, and HES4) were among the top 10 genes upregulated by both stimuli. Indeed, ALPL expression occurred concurrently with Notch activation and inhibiting Notch activity for up to 24 hours after BMP administration with DAPT (a gamma secretase inhibitor) completely abrogated hMSC osteoblastogenesis. Concordantly, RBPJ (recombination signal binding protein for immunoglobulin kappa J region, a critical downstream modulator of Notch signals) binding could be demonstrated within the ALPL and SP7 promoters. As such, siRNA‐mediated ablation of RBPJ decreased BMP‐mediated osteoblastogenesis. Finally, systemic Notch inhibition using diabenzazepine (DBZ) reduced BMP2‐induced calvarial bone healing in mice supporting the critical regulatory role of Notch signaling in BMP‐induced osteoblastogenesis.Bone morphogenetic protein (BMP) stimulation of bone‐marrow‐derived human mesenchymal progenitor cells (hMSCs) increases Notch proteins via increased Notch ligand Jagged1 expression. Canonical Notch signaling is required for BMP‐induced ALPL expression and osteoblastic commitment of hMSCs. Both BMP‐induced osteoblastogenesis and Notch‐induced osteoblastogenesis require Runx2.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162778/2/stem3245_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162778/1/stem3245.pd

Functional Characterization of EPDR1 as Novel Osteoblast Effector Gene at the BMD GWAS Implicated STARD3NL Locus

This talk addressed the discovery of EPDR1 as a novel human osteoblast effector genes using functional genomics approach of human BMD GWAS data. Other covered areas were the functional consequences of EPDR1 inactivation in human mesenchymal progenitor cell inflammation.University of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/191450/2/RFSS_120822_YW.pd

Variant-to-Gene Mapping and Functional Characterization of Novel Osteoblast Proteins

Plastic Surgery T32 fellows of University of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/191453/2/Variant-to-Gene_T32_042222.pd

Functional Characterization of Novel Osteoblast Effector Genes at the BMD-GWAS Implicated STARD3NL and DNM3 Loci

Spatial and Functional Genomics Seminar Series.Children's Hospital of Philadelphiahttp://deepblue.lib.umich.edu/bitstream/2027.42/191451/2/STARD3NL_DNM3OS_OCT2022.ppt

The intersection of Notch and BMP signaling during human osteoblast differentiation

This talk was designated to understand the cross-talk of BMP with Notch pathway in specification of human mesenchymal progenitor cells into osteoblasts.Notch Club, University of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/191452/2/NOTCHCLUB_FINAL_022618.ppt

C-Jun N-terminal Kinase (JNK) Isoform 2 Augments Human Osteoblast Differentiation by Integrating Bone Morphogenetic Protein Signaling and Canonical Notch Signaling

The c-Jun N-terminal kinases (JNK) are evolutionary conserved regulators of proliferation, differentiation, and cell death responses. In vivo studies have shown that Jnk1-/- and Jnk2-/- mice display varying degrees of osteopenia due to impaired bone formation. However, JNK1 and JNK2 isoform specific contribution to human osteoblast differentiation is lesser known. Here, we used small interfering RNA-mediated knockdown of JNK1 and JNK2 in bone-marrow derived human mesenchymal stem cells (hMSC) to evaluate their role in BMP-mediated osteoblast differentiation. Histochemical staining for alkaline phosphatase (ALP) expression and extracellular matrix mineralization using Alizarin red S showed that JNK2 activity is essential for hMSC osteoblastic differentiation, whereas JNK1 activity was dispensable. Gene expression analysis revealed a minimal effect on BMP-dependent RUNX2 and SP7 expression after JNK2 knock-down, but their levels were enhanced in JNK1 silenced cells. Since, BMP signaling requires canonical Notch signaling to promote human osteoblastogenesis, we evaluated JNK-isoform specific effects on Notch signaling components. The expression of Notch ligand JAG1 and downstream Notch target genes HES1 and NOTCH3 was reduced in response to BMP stimulation of JNK2 silenced cells, while the effects were modest after JNK1 knock-down. Consequently, the absolute requirement of JAG1 for BMP-mediated hMSC osteoblastogenesis was established as JAG1 silenced hMSC failed to enhance ALP expression and extracellular matrix mineralization. Finally, we generated JNK2-deficient immortalized hMSC using CRISPR-Cas9 genome editing and subjected these cells to BMP2 stimulation. Corresponding to the results with siRNA, JNK2 knock-out cells failed to produce extracellular matrix mineralization in response to BMP2. Collectively, these results establish a regulatory role of JNK2 in human osteoblast differentiation and suggest that it functions by integrating canonical BMP/SMAD signaling and Notch signaling in human cells.http://deepblue.lib.umich.edu/bitstream/2027.42/191455/2/JNKsiR kdh.ppt

Developing the Contextualized SERVQUAL Instrument for Measuring the Service Quality of Nepali Resorts: An Application of the Modified Delphi Technique

In resorts, service quality refers to the overall level of guest satisfaction during their stay. This includes a variety of factors, such as the friendliness and helpfulness of the staff, the cleanliness and condition of the rooms and common areas, the availability of amenities and activities, the efficiency of check-in and check-out procedures, and the resort’s overall atmosphere. To address above gap, this study aims to develop contextual service quality (SERVQUAL) instrument for resorts to obtain the consensus benchmark to measure the service quality of Nepali tourist standard resorts. The SERVQUQL is a measuring tool of service quality of different organizational settings, more importantly, in the hospitality and service sector. Therefore, to contextualize and align to SERVQUAL, the Modified Delphi Technique (MDT) was used to develop this instrument based on SERVQUAL theory. The instrument development was performed among 10 anonymous experts and stakeholders who have experience, knowledge, and expertise in the area of the hospitality and leisure industries. The findings identified that one more key construct is essential to realize the service quality of the tourist standard resorts. To carry out the mutual consensus among the experts and stakeholders, the interquartile range (IQR) value was taken from three points Likert scale to conduct the MDT from the second stage until the mutual consensus of experts and stakeholders. The IQR value was taken from each set of questions which was evaluated by the exports to obtain a consensus benchmark on the contextualized SERVQUQL construct and items. The data were presented using Microsoft Excel. In the final stage, 29 question statements out of 31 were found to have a high consensus benchmark among the experts and stakeholders. Therefore, 29 items from six constructs were accepted and included in the actual questionnaire to measure the service quality of Nepali tourist standard resorts. Our findings contribute to the SERVQUAL theory, practice, and future research