Cytoplasmic TSC-22 (transforming growth factor-beta-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.     

Collagen, type V, alpha1 (COL5A1) is regulated by TGF-beta in osteoblasts.

Bone matrix contains high concentrations of growth factors that are known to play important regulatory roles during osteogenesis, particularly transforming growth factor-beta (TGF-beta). Divergent effects of TGF-beta on bone formation have been reported both in vitro and in vivo depending upon experimental conditions, cells employed and their stage of maturation. In this study, we have used a clonal osteoblastic cell line MC3T3-E1, derived from newborn mouse calvaria, as an in vitro model of bone development. These cells undergo an ordered, time-dependent developmental sequence characterized by three stages (proliferation, differentiation and mineralization), over a 30-35-day period. In this study, cDNA microarray technology was used to study the expression profile of 8470 genes, in the presence of TGF-beta1 during osteoblast development. Microarray analysis revealed 120 cDNAs to be differentially expressed in MC3T3-E1 osteoblasts that had been treated with TGF-beta1. From the 120 differentially expressed genes, we selected Collagen, type V, alpha1 (COL5A1) {differential expression=+4.9} for further studies since it represents a previously uncharacterized component of the bone matrix. Using Northern blotting, we found that, when MC3T3-E1 cells were treated with TGF-beta1, COL5A1 was up-regulated during the proliferation and differentiation phases of osteogenesis. Furthermore, by a combination of RNA in situ hybridization and Northern blotting, we found COL5A1 mRNA to be expressed in the calvaria and developing bone of the E17.5 mouse embryos. Lastly, significant COL5A1 protein expression was observed by immunohistochemistry in the developing bone of the E17.5 mouse embryos. In conclusion, by the use of in vitro and in vivo approaches, we have discovered that the COL5A1 gene is a target of TGF-beta during osteogenesis.     

Role of TSC-22 during early embryogenesis in Xenopus laevis

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.     

Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1)

We recently identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene induced in adventitial fibroblasts after arterial injury. Cthrc1 is a 30 kDa secreted protein that has the ability to inhibit collagen matrix synthesis. Cthrc1 is also glycosylated and retains a signal sequence consistent with the presence of Cthrc1 in the extracellular space. In injured arteries and skin wounds, we have found Cthrc1 expression to be associated with myofibroblasts and sites of collagen matrix deposition. Furthermore, we demonstrated that Cthrc1 inhibits collagen matrix deposition in vitro. Using in situ hybridization and immunohistochemistry, we characterized the expression domains of Cthrc1 during murine embryonic development and in postnatal tissues. In mouse embryos, Cthrc1 was expressed in the visceral endoderm, notochord, neural tube, developing kidney, and heart. Abundant expression of Cthrc1 was observed in the developing skeleton, i.e., in cartilage primordia, in growth plate cartilage with exclusion of the hypertrophic zone, in the bone matrix and periostium. Bones from adults showed expression of Cthrc1 only in the bone matrix and periostium while the articular cartilage lacked expression. Cthrc1 is typically expressed at epithelial-mesenchymal interfaces that include the epidermis and dermis, basal corneal epithelium, airway epithelium, esophagus epithelium, choroid plexus epithelium, and meninges. In the adult kidney, collecting ducts and distal tubuli expressed Cthrc1. Collectively, the sites of Cthrc1 expression overlap considerably with those reported for TGF-beta family members and interstitial collagens. The present study provides useful information towards the understanding of potential Cthrc1 functions.     

Indian hedgehog in the late-phase differentiation in mouse chondrogenic EC cells, ATDC5: upregulation of type X collagen and osteoprotegerin ligand mRNAs

Endochondral bone formation includes a cascade of cellular events such as proliferation, maturation, hypertrophic conversion and calcification of chondrocytes and the cartilage replacement by bone. During these processes, hypertrophic conversion and calcification of chondrocytes (the late-phase differentiation) is a crucial process of chondrogenic differentiation. Indian hedgehog (Ihh), a secreted protein expressed in early hypertrophic chondrocytes, is thought to be involved in regulation of hypertrophic conversion via a feedback loop through the perichondrium. In the present study, we showed by Northern analysis and in situ hybridization that Smoothened (Smo), a key component in hedgehog signal transduction, was expressed in chondrocytes in both adult mice and mouse embryos at 16 days post-coitum in vivo, suggesting that Ihh directly acts on chondrocytes. We previously reported that Ihh, Patched and Smo were all expressed in differentiated ATDC5 cells. Exogenously administered mouse recombinant N-terminal protein of Ihh (mrIhh-N) upregulated the gene expression of type X collagen, a phenotypic marker of hypertrophic chondrocytes, as well as osteoprotegerin ligand (OPGL), a potent stimulator of osteoclastogenesis and osteoclast activity, while it did not modulate the expression of Ihh itself, bone morphogenetic protein (BMP)-4, BMP-6, transforming growth factor (TGF)-beta1 and TGF-beta2 in differentiated ATDC5 cells. Moreover, when added to the osteoclast cultures, mrIhh-N markedly stimulated the formation of resorption pits on dentine slices. Our data support the hypothesis that Ihh stimulated the late-phase chondrogenic differentiation in differentiated ATDC5 cells and upregulated the gene expression of OPGL in these cells.     

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation

Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor beta1 (TGF-beta1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF-beta1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-beta1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-beta1-induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-beta1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF-beta1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of TGF-beta1-induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-beta1 induced MC condensation.     

Expression screening for Lhx3 downstream genes identifies Thg-1pit as a novel mouse gene involved in pituitary development.

Thg-1pit, a novel mouse gene, was detected in a screen for genes that are differentially expressed in the developing pituitary of wild-type and Lhx3 null mutant embryos. The predicted translation product of the Thg-1pit gene contains a C-terminal TSC-box adjacent to a leucine zipper motif. These features are characteristic for the TSC-22/DIP/bun family of proteins. The onset of prominent Thg-1pit expression coincides with Lhx3 activation at early stages of pituitary development. Expression is further enhanced as cells begin to differentiate within the developing pituitary gland. No expression is observed in the pituitary rudiment of mutants that lack Lhx3 function. A possible role is thus suggested for Lhx3 activities in the regulation of Thg-1pit function during early steps of pituitary organogenesis.     

Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.     

Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.