Twist-ing cell fate: mechanistic insights into the role of twist in lineage specification/differentiation and tumorigenesis.

Bone marrow-derived mesenchymal stem cells (MSC), are multipotent cells that give rise to multiple lineages including osteoblasts, adipocytes, muscle, and fibroblasts. MSCs are useful for clinical applications such as cell therapy because they can be isolated from an individual and expanded for use in tissue repair, as well as other therapeutic applications, without immune rejection. However, one of the key problems in the use of MSCs for these applications is the efficiency of these cells to engraft and fully regenerate damaged tissues. Therefore, to optimize this process, a comprehensive understanding of the key regulators of MSCs self-renewal and maintenance are critical to the success of future cell therapy as well as other clinical applications. The basic helix loop helix transcription factor, Twist, plays a master regulatory role in all of these processes and, therefore, a thorough understanding of the mechanistic insights in the role of Twist in lineage specification/differentiation and tumorigenesis is vital to the success of future clinical applications for the therapeutic use of MSCs. In this article, we highlight the basic mechanisms and signaling pathways that are important to MSC fate, maintenance, and differentiation, as well as the critical role that Twist plays in these processes. In addition, we review the known literature suggesting a critical role for Twist in the generation of cancer stem cells, as this information may contribute to a broader understanding of stem cell biology and stem-cell-based therapeutics.

Snail knockdown reverses stemness and inhibits tumour growth in ovarian cancer.

To develop effective therapies for advanced high grade serous ovarian cancer (HGSOC), understanding mechanisms of recurrence and metastasis is necessary. In this study, we define the epithelial/mesenchymal status of cell lines that accurately model HGSOC, and evaluate the therapeutic potential of targeting Snai1 (Snail), a master regulator of the epithelial/mesenchymal transition (EMT) in vitro and in vivo. The ratio of Snail to E-cadherin (S/E index) at RNA and protein levels was correlated with mesenchymal morphology in four cell lines. The cell lines with high S/E index (OVCAR8 and COV318) showed more CSC-like, motile, and chemoresistant phenotypes than those with low S/E index (OVSAHO and Kuramochi). We tested the role of Snail in regulation of malignant phenotypes including stemness, cell motility, and chemotherapy resistance: shRNA-mediated knockdown of Snail reversed these malignant phenotypes. Interestingly, the expression of let-7 tumour suppressor miRNA was upregulated in Snail knockdown cells. Furthermore, knockdown of Snail decreased tumour burden in an orthotopic xenograft mouse model. We conclude that Snail is important in controlling HGSOC malignant phenotypes and suggest that the Snail/Let-7 axis may be an attractive target for HGSOC treatment.

Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells.

Although much is known about the hormonal regulation of osteoblastic cell differentiation, much less is known about the nuclear regulatory molecules that affect this process. We analyzed the expression of several regulatory molecules of the helix-loop-helix (H-L-H) group in primary mouse calvarial cells and in MC3T3-E1 mouse osteoblastic cells in situations representing different degrees of cellular differentiation. H-L-H class regulators are known to participate directly in directing cell fate and differentiation decisions in other mesodermal lineages. Two of the molecules that we studied, Id and E12, have well-established roles in this process. The other, mTwi, the murine homolog of the Drosophila twist gene, is a newly cloned mammalian H-L-H gene. Levels of E12 RNA remained unchanged during differentiation. On the other hand, in both primary osteoblastic cells and MC3T3-E1 cells, the abundance of Id and mTwi declined with cell maturation; mTwi less dramatically than Id. That Id expression is causally related to differentiation is suggested by the finding that MC3T3-E1 cells transfected with an Id-expression plasmid fail to undergo differentiation. We conclude that helix-loop-helix regulatory genes are expressed in mouse osteoblastic cells, where they are likely to participate in differentiation. The E12 gene product is likely to function as a positive modulating factor. In contrast, Id inhibits differentiation, probably by sequestering other H-L-H gene regulators, including E12, in inactive complexes. The precise role of mTwi is more speculative at this time, but the observed pattern of expression is consistent with a role in early and midmesodermal specification that is terminated as cells differentiate.

Human Dermo-1 has attributes similar to twist in early bone development.

Basic helix-loop-helix (bHLH) transcription factors are implicated in cell lineage determination and differentiation. Dermo-1 encodes a bHLH transcription factor that shares extensive homology with another bHLH transcription factor, Twist. We have cloned and characterized human Dermo-1 from two different bone cytoplasmic DNA (cDNA) libraries. Dermo-1 mRNA and protein expression were examined in human embryo and adult tissue sections. Dermo-1 is expressed in a subset of mesodermally and ectodermally derived tissues. We further examined expression of Dermo-1/Twist in human tissues and cell lines. In addition, we observed Dermo-1 expression in response to basic fibroblast growth factor in osteoblastic cell lines. To evaluate the functionality of the human Dermo-1 transcription factor in osteoblast metabolism, we made stable osteoblastic cell lines that over- and underexpress human Dermo-1. These cell lines were analyzed and compared with previously published data of similar cell lines transfected with Twist. Our results demonstrate that Dermo-1 caused changes similar to Twist in the osteogenic properties of osteoblastic cells, such as morphology, bone marker gene expression, and biochemical response to cytokines. However, Dermo-1 expression also has unique effects in regulating the mechanism of proliferation, on alkaline phosphatase enzyme activity, and in temporal expression patterns. We speculate that expression of Twist and Dermo-1 maintains cells in an osteoprogenitor or preosteoblast-like state, respectively, and prevents premature or ectopic osteoblast differentiation. Therefore, Twist and Dermo-1 must be sequentially downregulated in order to initiate the cascade of events responsible for osteogenic cell differentiation. These results indicate that, during osteoblast development, Dermo-1 may inhibit osteoblast maturation and maintain cells in a preosteoblast phenotype by utilizing mechanisms similar but not identical to those utilized by Twist.

Organ-specific transcripts of different size and abundance derive from the same pyruvate, orthophosphate dikinase gene in maize.

Analyses of genomic DNA and clones indicate that the pyruvate, orthophosphate dikinase (PPDK; ATP: pyruvate, orthophosphate phosphotransferase, EC 2.7.9.1) gene family of maize (Zea mays L. subsp. mays, line B73) contains two members. Restriction site and DNA sequence comparisons between PPDK genomic and leaf cDNA clones have revealed which gene encodes the isozyme involved in C4 photosynthesis. The region flanking the 5' end of this gene contains two 30-base-pair (bp) repetitive elements that may be involved in its light-regulated expression. Sequence analysis of genomic and leaf cDNA clones has also shown that the entire 7.3-kDa PPDK chloroplast transit peptide is encoded in the 436-bp first exon. Northern blot experiments with probes specific for the first exon and the 3' end of the gene showed that the smaller PPDK transcripts in roots and etiolated leaves [3.0 kilobases (kb) vs. the 3.5-kb green leaf transcript] lack the sequence encoding the chloroplast transit peptide. In addition, results from cDNA library screens have confirmed that the root transcript is approximately 50-fold less abundant than the green leaf transcript. Finally, sequence comparisons among cDNA clones from green leaves and roots and genomic clones representing both members of the PPDK gene family demonstrate that the green leaf transcript encoding the C4 isozyme and the root transcript are derived from the same gene.

Doxorubicin inhibits differentiation and enhances expression of the helix-loop-helix genes Id and mTwi in mouse osteoblastic cells.

Treatment with doxorubicin is associated with decreased levels of expression of muscle-specific genes in myocytes. This may be related to an effect on expression of helix-loop-helix (h-l-h) regulatory molecules since in myoblastic cells, doxorubicin inhibits Myo D expression and enhances Id expression. We have reported that expression of Id and mouse Twist (mTwi), another h-l-h molecule, decline in association with differentiation in osteoblastic cells. We have sought, therefore, to determine the effect of doxorubicin on MC-3T3-E1 osteoblastic cells. Treatment with doxorubicin decreased total cellular protein content, reduced [3H]-leucine incorporation into protein, inhibited proliferation and diminished alkaline phosphatase activity. Glucose utilization and lactate production were not adversely affected. Id expression was increased by doxorubicin treatment under growth conditions but not differentiation conditions. Expression of mTwi was markedly increased under both growth and differentiation conditions. These data support the contention that Id and mTwi may regulate differentiation in osteoblastic cells.

Transcriptional regulation of alpha 2(I) collagen gene expression by fibroblast growth factor-2 in MC3T3-E1 osteoblast-like cells.

Fibroblast growth factor-2 (FGF-2) stimulates proliferation and inhibits differentiated function of osteoblasts by suppressing synthesis of type I collagen and other proteins. However, little is known regarding the molecular mechanisms regulating the suppressive effects of FGF-2 on type I collagen synthesis in osteoblasts. The zinc finger transcription factor Egr-1 and the basic helix-loop-helix (bHLH) family of proteins have been implicated in the regulation of genes crucial to mesodermal cell growth and differentiation. The aim of this study was to determine whether Egr-1 and TWIST might be potential transcriptional regulators of the inhibitory effects of FGF-2 on alpha2(I) collagen expression in MC3T3-E1 osteoblasts which undergo a developmental sequence in vitro. Upon treatment of undifferentiated MC3T3-E1 cells with 1 nM FGF-2, Egr-1 mRNA increased with the effect maximal after 30-60 min. TWIST mRNA also increased with the effect maximal at 2 h. We analyzed the transcriptional control of alpha2(I) collagen gene expression by FGF-2 by transient transfection of an alpha2(I) collagen-luciferase construct (pH5) into undifferentiated MC3T3-E1 cells. The activity of the pH5 luciferase promoter decreased in a dose-dependent manner following treatment with.01 and 1 nM FGF-2. We identified putative Egr-1 and TWIST recognition sequences in the proximal region of the promoter for the murine alpha2(I) collagen gene and a putative Egr-1 site in the 5' region of the murine TWIST promoter. In gel mobility shift assays, potential Egr-1 response elements in the 5' region of the murine TWIST and alpha2(I) collagen genes demonstrated specific Egr-1 binding activity with bFGF-treated nuclear extracts obtained from MC3T3-E1 cells. These results indicate that Egr-1 and TWIST are expressed in undifferentiated MC3T3-E1 osteoblast-like cells following treatment with FGF-2 and they may be potential transcriptional regulators of FGF-2s negative effects on alpha2(I) collagen gene expression. J. Cell. Biochem. 80:550-559, 2001. Published 2001 Wiley-Liss, Inc.

Endochondral and intramembranous fetal bone development: osteoblastic cell proliferation, and expression of alkaline phosphatase, m-twist, and histone H4.

We have previously studied the expression of alkaline phosphatase (ALP) and alpha2(I) collagen (two phenotypic markers of osteoblastic cell differentiation) during development of the rat mandible, and the spatial and temporal distribution of the respective transcripts. Our current studies utilize the rat mandible and hind foot as in vivo model systems to investigate the relationship between osteoblastic differentiation and proliferation during intramembranous and endochondral bone formation. Pregnant rats, at 15.17, and 19 days of gestation were intraperitoneally injected with various doses of [3H]-thymidine, and sacrificed at various time intervals in order to label dividing embryonic osteoblastic and preosteoblastic cells. Cross sections through the mid-body of 15-day embryos showed [3H-thymidine dose-dependent labeling of a relatively high percentage of cells in the liver (49 +/- 8% at 440 muCi) and a lower percentage of cells of the developing vertebral cartilage (29 +/- 6% at 440 muCi). ALP-positive condensed mesenchyme--consisting of mandibular preosteoblast (15 days of gestation) showed a relatively high (32 +/- 5%) level of [3H]-thymidine labeling, compared to surrounding ALP-negative loose mesenchymal cells (22 +/- 1%). Similar results were observed in the developing hind foot of 19-day embryos for ALP-positive cells (15 +/- 6%) and surrounding ALP-negative cells (13 +/- 5%). In both the hind foot and the mandible an overall decrease in labeling was observed during bone development. RNA samples from these tissues show increasing amounts of ALP mRNA, and decreasing amounts of histone H4 mRNA between days 15 and 19 of gestation. These data indicate that a general inverse correlation between osteoblastic differentiation and proliferation, similar to the correlation previously described in cultured osteogenic cells, is also present in developing bones in vivo. However, these results indicate that ALP-positive preosteoblasts, committed to the osteoblastic lineage, maintain their proliferative capacity. In an attempt to elucidate underlying molecular mechanisms, we further investigated the levels of expression of m-twist in these tissues. This member of the basic helix-loop-helix family of transcription regulators has been previously implied as playing a role in osteoblast differentiation in culture. Our results demonstrate a decrease in m-twist levels during bone development in both the mandible and the hind foot.

Insulin-like growth factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor.

In this study we investigated the molecular mechanisms whereby insulin-like growth factor 1 (IGF-1) induced Twist gene expression and the role of Twist in the anti-apoptotic actions of the IGF-1 receptor. In NIH-3T3 fibroblasts overexpressing the human IGF-1 receptor (NWTb3), treatment with IGF-1 (10(-8) m) for 1 and 4 h increased the level of Twist mRNA as well as protein by 3-fold. In contrast, insulin at physiological concentrations did not stimulate Twist expression in NIH-3T3 fibroblasts overexpressing the human insulin receptor. The IGF-1 effect was specific for the IGF-1 receptor since, in cells overexpressing a dominant negative IGF-1 receptor, IGF-1 failed to increase Twist expression. Pre-incubation with the ERK1/2 inhibitor U0126 or expression of a dominant negative MEK-1 abolished the effect of IGF-1 on Twist mRNA expression in NWTb3 cells, suggesting that Twist induction by IGF-1 occurs via the mitogen-activated protein kinase signaling pathway. In vivo, IGF-1 injection increased the mRNA level of Twist in mouse skeletal muscle, the major site of Twist expression. Finally, using an antisense strategy, we demonstrated that a reduction of 40% in Twist expression decreased significantly the ability of IGF-1 to rescue NWTb3 cells from etoposide-induced apoptosis. Taken together, these results define Twist as an important factor involved in the anti-apoptotic actions of the IGF-1 receptor.

Bone morphogenetic protein inhibits differentiation and affects expression of helix-loop-helix regulatory molecules in myoblastic cells.

Bone morphogenetic protein (BMP) reproducibly induces chondrogenesis and osteogenesis when implanted into skeletal muscle. The exact identity of the cell that responds to BMP is not known. Furthermore, controversy exists regarding the possibility that myoblastic cells may transdifferentiate to chondrocytes and osteoblasts under the influence of BMP. We have therefore, undertaken studies on the effects of BMP on differentiation in L6 and C2C12 cells, two rodent myoblastic cell lines. To gain insights into the mechanisms of action of BMP, we have studied the effects of BMP on the levels of expression of the four known myogenic determination genes: myogenin, Myo D, herculin, and myf-5. BMP inhibited myogenesis in myoblastic cells. Convincing evidence of transdifferentiation of myoblasts to chondrocytes or osteoblasts was not seen. BMP inhibited the expression of all four myogenic determination genes.

Genomic and cDNA clones for maize phosphoenolpyruvate carboxylase and pyruvate,orthophosphate dikinase: Expression of different gene-family members in leaves and roots.

We have isolated cDNA clones for the maize leaf enzymes phosphoenolpyruvate (P-ePrv) carboxylase [orthophosphate:oxaloacetate carboxy-lyase (phosphorylating) EC 4.1.1.31] and pyruvate,orthophosphate (Prv,P(i)) dikinase (ATP:pyruvate,orthophosphate phosphotransferase, EC 2.7.9.1) by exploiting the light-inducibility and large size of the mRNAs (3.5 kilobases) that encode the two enzymes. The clones were identified by hybrid-selection and immunoprecipitation assays. From a maize genomic library, two different types of genomic clones were screened with both the P-ePrv carboxylase and the Prv,P(i) dikinase cDNA clones. Information from these genomic clones and genome blots indicates that the P-ePrv carboxylase gene family has at least three members and the Prv,P(i) dikinase family at least two. Transcripts for both enzymes were detected in green leaves, etiolated leaves, and roots. The results show that the P-ePrv carboxylase mRNAs in green leaves and roots are encoded by different genes. Whereas the P-ePrv carboxylase mRNAs in all three tissues appear to be the same size, the Prv,P(i) dikinase mRNA in green leaves is about 0.5 kilobases longer than the Prv,P(i) dikinase mRNAs in etiolated leaves and roots. It is possible that all these Prv,P(i) dikinase transcripts are encoded by one gene, and the size differences may correspond to the presence or absence of a sequence encoding a chloroplast transit peptide.

TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage.

Basic helix-loop-helix (bHLH) transcription factors have been shown to play an important role in controlling cell type determination and differentiation. TWIST, a member of the bHLH transcription factor family, is involved in the development of mesodermally derived tissue, including the skeleton. We examined the role of human TWIST in osteoblast metabolism using stable expression of sense and antisense TWIST in human osteoblast HSaOS-2 cells. Changes in morphology and osteogenic phenotype characterized these stable clones. Cells that overexpressed TWIST exhibited a spindle shaped morphology, reduced levels of alkaline phosphatase, a reduced proliferation rate, and failed to respond to basic fibroblast growth factor (bFGF). In contrast, those that underexpressed TWIST demonstrated a cuboidal epithelial-like morphology characteristic of differentiated osteoblasts. TWIST antisense cells exhibited increased levels of alkaline phosphatase and type I collagen mRNA, initiated osteopontin mRNA expression, and had a reduced proliferation rate. These results indicate that TWIST overexpressing cells may de-differentiate and remain in an osteoprogenitor-like state, and antisense TWIST cells progress to a more differentiated mature osteoblast-like state. Therefore, the level of TWIST can influence osteogenic gene expression and may act as a master switch in initiating bone cell differentiation by regulating the osteogenic cell lineage.