Uniaxial mechanical tension promoted osteogenic differentiation of rat tendon-derived stem cells (rTDSCs) via the Wnt5a-RhoA pathway

Chronic tendinopathy is a tendon disorder that is common in athletes and individuals whose tendons are subjected to repetitive strain injuries. The presence of ossification worsened the clinical manifestation of the disorder. The change of tendon loading due to mechanical overload, compression, or disuse have been implicated as the possible etiologies, but the pathological mechanisms of tendinopathy remain unclear. In this study, we demonstrated that ossification in tendon tissue might be due to the osteogenesis of tendon‐derived stem cells (TDSCs) induced by uniaxial mechanical tension (UMT) which mimics the mechanical loading in tendon. Rat TDSCs (rTDSCs) could be induced to differentiate into osteogenic lineage after treatment with 2% elongation UMT for 3 days as shown by the increased expression Runx2 mRNA and protein, Alpl mRNA, collagen type 1 alpha 1 (Col1a1) mRNA, ALP activity, and ALP cytochemical staining. RhoA, an osteogenesis regulator, was activated in rTDSCs upon UMT stimulation. Blockage of RhoA activity in rTDSCs by C3 toxin or ROCK activity, a downstream target of RhoA, by Y‐27632 inhibited UMT‐induced osteogenesis in rTDSCs. UMT up‐regulated the mRNA expression of Wnt5a but not the other non‐canonical Wnts. The inhibition of Wnt5a expression by siRNA abolished UMT‐induced Runx2 mRNA expression and RhoA activation in rTDSCs and the inhibition of Runx2 expression could be rescued by addition of LPA, a RhoA activator. In conclusion, our results showed that UMT induced osteogenic differentiation of rTDSCs via the Wnt5a‐RhoA pathway, which might contribute to ectopic ossification in tendon tissue due to mechanical loading. J. Cell. Biochem. 113: 3133–3142, 2012. © 2012 Wiley Periodicals, Inc.     

Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis

Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a ‘progression network’ that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.     

Multilineage differentiation of dental follicle cells and the roles of Runx2 over‐expression in enhancing osteoblast/cementoblast‐related gene expression in dental follicle cells

Objectives: Dental follicle cells (DFCs) provide the origin of periodontal tissues, and Runx2 is essential for bone formation and tooth development. In this study, pluripotency of DFCs was evaluated and effects of Runx2 on them were investigated. Materials and methods: The DFCs were induced to differentiate towards osteoblasts, adipocytes or chondrocytes, and alizarin red staining, oil red O staining or alcian blue staining was performed to reveal the differentiated states. Bone marrow stromal cells (BMSCs) and primary mouse fibroblasts served as controls. DFCs were also infected with recombinant retroviruses encoding either full‐length Runx2 or mutant Runx2 without the VWRPY motif. Western blot analysis, real‐time real time RT‐PCR and in vitro mineralization assay were performed to evaluate the effects of full‐length Runx2 or mutant Runx2 on osteogenic/cementogenic differentiation of the cells. Results: The above‐mentioned staining methods demonstrated that DFCs were successfully induced to differentiate towards osteoblasts, adipocytes or chondrocytes respectively, confirming the existence of pluripotent mesenchymal stem cells in dental follicle tissues. However, staining intensity in DFC cultures was weaker than in BMSC cultures. Real‐time PCR analysis indicated that mutant Runx2 induced a more pronounced increase in expression levels of OC, OPN, Col I and CP23 than full‐length Runx2. Mineralization assay also showed that mutant Runx2 increased mineralization nodule formation more prominently than full‐length Runx2. Conclusions: Multipotent DFCs can be induced to differentiate towards osteoblasts, adipocytes or chondrocytes in vitro. Runx2 over‐expression up‐regulated expression levels of osteoblast/cementoblast‐related genes and in vitro enhanced osteogenic differentiation of DFCs. In addition, mutant Runx2‐induced changes in DFCs were more prominent than those induced by full‐length Runx2.     

Sphingolipid long chain base phosphates can regulate apoptotic-like programmed cell death in plants

Sphingolipids are ubiquitous components of eukaryotic cells and sphingolipid metabolites, such as the long chain base phosphate (LCB-P), sphingosine 1 phosphate (S1P) and ceramide (Cer) are important regulators of apoptosis in animal cells. This study evaluated the role of LCB-Ps in regulating apoptotic-like programmed cell death (AL-PCD) in plant cells using commercially available S1P as a tool. Arabidopsis cell cultures were exposed to a diverse array of cell death-inducing treatments (including Cer) in the presence of S1P. Rates of AL-PCD and cell survival were recorded using vital stains and morphological markers of AL-PCD. Internal LCB-P levels were altered in suspension cultured cells using inhibitors of sphingosine kinase and changes in rates of death in response to heat stress were evaluated. S1P reduced AL-PCD and promoted cell survival in cells subjected to a range of stresses. Treatments with inhibitors of sphingosine kinase lowered the temperature which induced maximal AL-PCD in cell cultures. The data supports the existence of a sphingolipid rheostat involved in controlling cell fate in Arabidopsis cells and that sphingolipid regulation of cell death may be a shared feature of both animal apoptosis and plant AL-PCD.     

Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis

Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphorylated by the actions of two S1P-specific phosphatases, sphingosine-1-phosphate phosphatases 1 and 2. To identify the physiological functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1^−/− mice appeared normal at birth, but during the 1st week of life they exhibited stunted growth and suffered desquamation, with most dying before weaning. Both Sgpp1^−/− pups and surviving adults exhibited multiple epidermal abnormalities. Interestingly, the epidermal permeability barrier developed normally during embryogenesis in Sgpp1^−/− mice. Keratinocytes isolated from the skin of Sgpp1^−/− pups had increased intracellular S1P levels and displayed a gene expression profile that indicated overexpression of genes associated with keratinocyte differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis.     

A novel RUNX2 mutation in exon 8, G462X,in a patient with Cleidocranial Dysplasia

To identify a novel mutation of Runx2 gene in Cleidocranial Dysplasia (CCD) patients and to characterize the functional consequences of this mutation. The subjects consisted of 12 Korean CCD patients. After oral epithelial cells were collected using a mouthwash technique, genomic DNA was extracted. Screening for Runx2 mutation was performed using direct sequencing of polymerase chain reaction (PCR) products for exons 1‐8. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transacting activity, cyclohexamide chase assay for Runx2 protein stability, real‐time PCR for mRNA level of Runx2 downstream bone marker genes, and alkaline phosphatase (ALP) staining assay in mesenchymal stem cells for osteoblast differentiation. Of the 12 patients, seven showed Runx2 mutations reported previously and four showed no mutation. A novel mutation, G462X in exon 8, which was located in the C‐terminus of proline/serine/threonine‐rich (PST) domain, was found in one patient. In the luciferase assay, Runx2 transacting activity was decreased in Runx2‐G462X transfected cells. In the cyclohexamide chase assay, Runx2‐G462X mutation reduced the stability of Runx2 protein. Expression of the bone marker genes (osteocalcin, ALP, Type I collagen αI, matrix metalloproteinase‐13, bone sialoprotein, and osteopontin) decreased in G462X‐transfected cells. In the ALP staining assay, osteoblast differentiation was reduced in Runx2‐G462X overexpressed cell. The G462X mutation might reduce the Runx2 transacting activity, lower the protein stability, downgrade the expression of bone marker genes, and eventually diminish osteoblast differentiation in CCD patients.     

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G(2) /M phase arrest

Objectives: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti‐cancer treatment have been sought from natural resources. Here, we have investigated anti‐proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells. Material and methods: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate levels were analysed by HPLC. Results: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G₂/M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21^waf1/cip1 was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate in myriocin‐treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells. Conclusions: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21^waf1/cip1, followed by inhibition of cyclin B1 and cdc2, resulting in G₂/M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism‐based therapy for this type of skin cancer.