Fatigue-induced Orosomucoid 1 Acts on C-C Chemokine Receptor Type 5 to Enhance Muscle Endurance.

Understanding and managing fatigue is a significant challenge in clinic and society. In attempting to explore how the body responds to and regulates fatigue, we found in rodent fatigue models that orosomucoid 1 (ORM1) was significantly increased in multiple tissues, including blood and muscle. Interestingly, administration of exogenous ORM1 increased muscle glycogen and enhanced muscle endurance, whereas ORM1 deficiency resulted in a significant decrease of muscle endurance both in vivo and in vitro, which could largely be restored by exogenous ORM1. Further studies demonstrated that ORM1 can bind to C-C chemokine receptor type 5 (CCR5) on muscle cells and deletion of the receptor abolished the effect of ORM1. Thus, fatigue upregulates the level of ORM1, which in turn functions as an anti-fatigue protein to enhance muscle endurance via the CCR5 pathway. Modulation of the level of ORM1 and CCR5 signaling could be a novel strategy for the management of fatigue.

Infiltration of mesenchymal stem cells into PEGDA hydrogel.

BACKGROUND: Attachment of cells to fully hydrated hydrogel biomaterials, such as PEGDA, has proven challenging because of the hydrophobic cellular membrane. OBJECTIVE: To demonstrate undifferentiated human mesenchymal stem cells (hMSCs) and hMSC-derived chondrocytes infiltrated and attached to unmodified PEGDA hydrogel. METHODS: Human MSCs and MSC-Cys were cultured in and on PEGDA hydrogel. Attachment was verified by SEM and confocal microscopy and was accompanied by vinculin expression, indicating the presence of focal contacts. Infiltration was confirmed by H&E and fluorescence staining. RESULTS: Cells cultured on top of PEGDA hydrogel infiltrated the material on the order of micrometers. CONCLUSIONS: These findings will aid in understanding the cell-scaffold interaction for regenerative medicine constructs.

The development of Wilms tumor: from WT1 and microRNA to animal models.

Wilms tumor recapitulates the development of the kidney and represents a unique opportunity to understand the relationship between normal and tumor development. This has been illustrated by the findings that mutations of Wnt/ß-catenin pathway-related WT1, ß-catenin, and WTX together account for about one-third of Wilms tumor cases. While intense efforts are being made to explore the genetic basis of the other two-thirds of tumor cases, it is worth noting that, epigenetic changes, particularly the loss of imprinting of the DNA region encoding the major fetal growth factor IGF2, which results in its biallelic over-expression, are closely associated with the development of many Wilms tumors. Recent investigations also revealed that mutations of Drosha and Dicer, the RNases required for miRNA generation, and Dis3L2, the 3'-5' exonuclease that normally degrades miRNAs and mRNAs, could cause predisposition to Wilms tumors, demonstrating that miRNA can play a pivotal role in Wilms tumor development. Interestingly, Lin28, a direct target of miRNA let-7 and potent regulator of stem cell self-renewal and differentiation, is significantly elevated in some Wilms tumors, and enforced expression of Lin28 during kidney development could induce Wilms tumor. With the success in establishing mice nephroblastoma models through over-expressing IGF2 and deleting WT1, and advances in understanding the ENU-induced rat model, we are now able to explore the molecular and cellular mechanisms induced by these genetic, epigenetic, and miRNA alterations in animal models to understand the development of Wilms tumor. These animal models may also serve as valuable systems to assess new treatment targets and strategies for Wilms tumor.

A transcribed pseudogene of MYLK promotes cell proliferation.

Pseudogenes are considered nonfunctional genomic artifacts of catastrophic pathways. Recent evidence, however, indicates novel roles for pseudogenes as regulators of gene expression. We tested the functionality of myosin light chain kinase pseudogene (MYLKP1) in human cells and tissues by RT-PCR, promoter activity, and cell proliferation assays. MYLKP1 is partially duplicated from the original MYLK gene that encodes nonmuscle and smooth muscle myosin light chain kinase (smMLCK) isoforms and regulates cell contractility and cytokinesis. Despite strong homology with the smMLCK promoter (∼ 89.9%), the MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. Moreover, MYLKP1 and smMLCK exhibit negatively correlated transcriptional patterns in normal and cancer cells with MYLKP1 strongly expressed in cancer cells and smMLCK highly expressed in non-neoplastic cells. For instance, expression of smMLCK decreased (19.5 ± 4.7 fold) in colon carcinoma tissues compared to normal colon tissues. Mechanistically, MYLKP1 overexpression inhibits smMLCK expression in cancer cells by decreasing RNA stability, leading to increased cell proliferation. These studies provide strong evidence for the functional involvement of pseudogenes in carcinogenesis and suggest MYLKP1 as a potential novel diagnostic or therapeutic target in human cancers.

Shear stress induces osteogenic differentiation of human mesenchymal stem cells.

AIM: To determine whether fluid flow-induced shear stress affects the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) into osteogenic cells. MATERIALS & METHODS: hMSCs cultured with or without osteogenic differentiation medium were exposed to fluid flow-induced shear stress and analyzed for alkaline phosphatase activity and expression of osteogenic genes. RESULTS: Immediately following shear stress, alkaline phosphatase activity in osteogenic medium was significantly increased. At days 4 and 8 of culture the mRNA expression of bone morphogenetic protein-2 and osteopontin was significantly higher in hMSCs subjected to shear stress than those cultured in static conditions. However, hMSCs cultured in osteogenic differentiation medium were less responsive in gene expression of alkaline phosphatase and bone morphogenetic protein-2. CONCLUSION: These data demonstrate that shear stress stimulates hMSCs towards an osteoblastic phenotype in the absence of chemical induction, suggesting that certain mechanical stresses may serve as an alternative to chemical stimulation of stem cell differentiation.

Cytoskeletal changes of mesenchymal stem cells during differentiation.

Mesenchymal stem cells (MSCs) are progenitors for tissues such as bone and cartilage. In this report, the actin cytoskeleton and nanomechanobiology of human mesenchymal stem cells (hMSCs) were studied using fluorescence microscopy and atomic force microscopy (AFM). Human MSCs were differentiated into chondrocytes and osteoblasts as per previous approaches. Cytochalasin D (CytD) was used to temporarily disrupt cytoskeleton in hMSCs, hMSC-chondrocytes (hMSC-Cys) and hMSC-osteoblasts (hMSC-Obs). Fluorescence microscopy revealed a dose-dependent response to CytD. Removal of CytD from the media of cytoskeleton-disrupted cells led to the recovery of the cytoskeletal structures, as confirmed by both fluorescence microscopy and AFM. Force-volume imaging by AFM evaluated the nanomechanics of all three cell types before, during, and after CytD treatment. Cytochalasin D disruption of cytoskeleton had marked effects on hMSCs and hMSC-Cys, in comparison with limited cytoskeleton disruption in hMSC-Obs, as confirmed qualitatively by fluorescence microscopy and quantitatively by AFM. Treatment with CytD resulted in morphology changes of all cell types, with significant decreases in the observed Young's Moduli of hMSCs and hMSC-Cys. These data suggest human mesenchymal stem cells alter their cytoskeletal components during differentiation. Additional studies will address the mechanisms of cytoskeletal changes using biochemical and biophysical methods.