Differentiation of human skeletal muscle stem cells into odontoblasts is dependent on induction of α1 integrin expression.

Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7 integrin-positive human skeletal muscle stem cells (α7(+)hSMSCs) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS-harvested α7(+)hSMSCs were treated in suspension with retinoic acid followed by culture on a gelatin scaffold in the presence of BMP-4. Following this protocol, α7(+)hSMSCs were induced to down-regulate myogenic genes (MYOD and α7 integrin) and up-regulate odontogenic markers including dentin sialophosphoprotein, matrix metalloproteinase-20 (enamelysin), dentin sialoprotein, and alkaline phosphatase but not osteoblastic genes (osteopontin and osteocalcin). Following retinoic acid and gelatin scaffold/BMP-4 treatment, there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1ß1 integrin was strongly up-regulated with the attenuation of muscle-specific α7ß1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers dentin sialophosphoprotein and matrix metalloproteinase-20. These results strongly suggest that the differentiation of α7(+)hSMSCs along the odontogenic lineage is dependent on the concurrent expression of α1 integrin.

Transcriptional profiling identifies upregulated genes following induction of epithelial-mesenchymal transition in squamous carcinoma cells.

During the progression of head and neck squamous cell carcinoma (HNSCC), the induction of an epithelial-mesenchymal transition (EMT) program may play a critical role in the dissemination of cells from the primary tumor to distant metastatic foci. The process of EMT involves the activation of several important genes and pathways to help maintain survival and growth and evolve into highly invasive and metastatic variants. In this study, expression microarray analysis identified a set of 145 upregulated genes in EMT-like HNSCC cells. Some of the strongly upregulated transcripts include genes that are reportedly involved in invasion and metastasis, such as DOCK10, LOX, ROBO1 and SRGN. Importantly, the Tbx3 gene, a member of the T-box transcription factor, was strongly upregulated in SCC cells displaying an EMT-like phenotype compared to cells with an epitheloid, non-EMT behavior. Tbx3 was also found to be strongly upregulated at the protein and gene expression level in an experimental model of snail-induced EMT cells. In addition, siRNA-induced Tbx3 depletion modestly suppressed cell invasion while enhancing Tbx3-mediated resistance to anoikis. Our findings provide evidence that Tbx3 overexpression promotes SCC cell survival displaying an EMT phenotype. This set of newly identified genes that are modulated during EMT-like conversion may be important diagnostic biomarkers during the process of HNSCC progression.

Bcl-2 overexpression induces a partial epithelial to mesenchymal transition and promotes squamous carcinoma cell invasion and metastasis.

Evidence shows that Bcl-2 family members play a direct role in the development of some human malignancies. However, the mechanism by which Bcl-2 may influence tumor cell invasion and metastasis remains unclear. Ectopic overexpression of Bcl-2 in the human squamous carcinoma cell line HSC-3 enhanced tumorigenicity and experimental pulmonary metastasis. Interestingly, Bcl-2-expressing cells showed morphologic changes that resembled that of cells with an epithelial-mesenchymal transition phenotype. Analysis revealed increased N-cadherin and vimentin expression in parallel with attenuated E-cadherin level, along with enhanced migration and invasive behavior. Zymography studies confirmed elevated levels of matrix metalloproteinase-9 (MMP-9) in media of Bcl-2-expressing cells. siRNA-mediated suppression of N-cadherin expression not only prevented the enhanced invasion but also blocked the increased MMP-9 expression induced by elevated Bcl-2 expression. Accordingly, pharmacologic inhibition of MMP-9 abrogated the increased tumor cell invasion. Furthermore, the Bcl-2-mediated increase in MMP-9 expression and tumor cell invasion was dependent on fibroblast growth factor receptor-1 or extracellular signal-regulated kinase signaling. Collectively, the data establish that Bcl-2 overexpression in squamous carcinoma cells induces a partial epithelial to mesenchymal transition that promotes not only survival but also invasion and metastasis through the N-cadherin/fibroblast growth factor receptor/extracellular signal-regulated kinase pathway.

Sdc1 negatively modulates carcinoma cell motility and invasion.

During cancer progression, tumor cells eventually invade the surrounding collagen-rich extracellular matrix. Here we show that squamous cell carcinoma cells strongly adhere to Type I collagen substrates but display limited motility and invasion on collagen barriers. Further analysis revealed that in addition to the alpha2beta1 integrin, a second collagen receptor was identified as Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan. We demonstrate that siRNA-mediated depletion of Sdc1 reduced adhesion efficiency to collagen I, whereas knockdown of Sdc4 was without effect. Importantly, silencing Sdc1 expression caused reduced focal adhesion plaque formation and enhanced cell spreading and motility on collagen I substrates, but did not alter cell motility on other ECM substrates. Sdc1 depletion ablated adhesion-induced RhoA activation. In contrast, Rac1 was strongly activated following Sdc1 knockdown, suggesting that Sdc1 may mediate the link between integrin-induced actin remodeling and motility. Taken together, these data substantiate the existence of a co-adhesion receptor system in tumor cells, whereby Sdc1 functions as a key regulator of cell motility and cell invasion by modulating RhoA and Rac activity. Downregulation of Sdc1 expression during carcinoma progression may represent a mechanism by which tumor cells become more invasive and metastatic.

Analysis of human muscle stem cells reveals a differentiation-resistant progenitor cell population expressing Pax7 capable of self-renewal.

Studies using mouse models have established a critical role for resident satellite stem cells in skeletal muscle development and regeneration, but little is known about this paradigm in human muscle. Here, using human muscle stem cells, we address their lineage progression, differentiation, migration, and self-renewal. Isolated human satellite cells expressed alpha7-integrin and other definitive muscle markers, were highly motile on laminin substrates and could undergo efficient myotube differentiation and myofibrillogenesis. However, only a subpopulation of the myoblasts expressed Pax7 and displayed a variable lineage progression as measured by desmin and MyoD expression. Analysis identified a differentiation-resistant progenitor cell population that was Pax7+/desmin- and capable of self-renewal. This study extends our understanding of the role of Pax7 in regulating human satellite stem cell differentiation and self-renewal.

STAT3 signaling is induced by intercellular adhesion in squamous cell carcinoma cells.

The signal transducer and activator of transcription-3 (STAT3) frequently activated during tumor progression has been linked to enhanced cell growth. In squamous cell carcinoma of the head and neck (HNSCC), STAT3 signaling has been shown to inhibit apoptosis and induce a more aggressive phenotype through the activation of specific signaling pathways. In the present study, we have examined the potential mechanism by which cell-cell contact initiates STAT3 activation. Using a panel of HNSCC cell lines, Ca(+2)-dependent cell-cell adhesion and adherens junction formation in multicellular aggregates triggered phosphorylation of STAT3-Y705 and STAT1-Y701. This intercellular adhesion-induced STAT3 activation was mediated by JAK and Src signaling and partially by EGFR signaling. In addition, immunolocalization studies revealed initial formation of phosphorylated STAT3-Y705 at nascent E-cadherin cell junctions with eventual translocation to the nucleus in cell aggregates. Adhesion-mediated STAT activation in monolayer and cell aggregate cultures required functional E-cadherin. These results indicate that, in HNSCC cells, cadherin-mediated intercellular adhesion induces STAT signaling that may modulate cell survival and resistance to apoptosis during tumor progression.

Modulation of satellite cell adhesion and motility following BMP2-induced differentiation to osteoblast lineage.

Quiescent satellite cells represent pluripotent stem cells capable of differentiating into other lineages. To define the potential changes in adhesion and motility in these differentiating cells, we utilized an established model system of murine-derived satellite cells induced with BMP2 to undergo osteoblastic differentiation. When mouse myogenic satellite cells were treated with BMP2, myogenesis was inhibited, and interaction with extracellular matrix ligands was altered. alpha7 integrin expression was rapidly downregulated with attenuation of adhesion and migration on laminin substrates. BMP2 also induced alpha2 integrin expression with increased adhesion and motility on collagen substrates as the pluripotent myoblasts develop into the osteogenic lineage. We examined the effect of BMP2 on alpha7 promoter activity in myoblasts using a CAT reporter gene. BMP2 was found to suppress integrin expression through a transcriptional mechanism. The results identify a novel role for BMP2 in modulating satellite cell integrin expression and altering their interactions with the microenvironment during osteoblastic differentiation.

alpha7 integrin expressing human fetal myogenic progenitors have stem cell-like properties and are capable of osteogenic differentiation.

During muscle development, precursor cells fuse to form myofibers. Following injury in adult muscle, quiescent satellite cells become activated to regenerate muscle in a fashion similar to fetal development. Recent studies indicate that murine skeletal myoblasts can differentiate along multiple cell lineages including the osteoblastic pathway. However, little is known about the multipotency of human myogenic cells. Here, we isolate myogenic precursor cells from human fetal and adult muscle by sorting for the laminin-binding alpha7 integrin and demonstrate their differentiation potential and alteration in adhesive behavior. The alpha7-positive human fetal progenitors were efficient at forming myotubes and a majority expressed known muscle markers including M-cadherin and c-Met, but were heterogeneous for desmin and MyoD expression. To test their pluripotent differentiation potential, enriched populations of alpha7-positive fetal cells were subjected to inductive protocols. Although the myoblasts appeared committed to a muscle lineage, they could be converted to differentiate along the osteoblastic pathway in the presence of BMP-2. Interestingly, osteogenic cells showed altered adhesion and migratory activity that reflected growth factor-induced changes in integrin expression. These results indicate that alpha7-expressing fetal myoblasts are capable of differentiation to osteoblast lineage with a coordinated switch in integrin profiles and may represent a mechanism that promotes homing and recruitment of myogenic stem cells for tissue repair and remodeling.

Alpha7 integrin expression is negatively regulated by deltaEF1 during skeletal myogenesis.

Alpha7 integrin levels increase dramatically as myoblasts differentiate to myotubes. A negative regulatory element with putative sites for deltaEF1 is present in the alpha7 proximal promoter region. To define the role of deltaEF1 in regulating alpha7 integrin expression, we overexpressed deltaEF1 in C2C12 myoblasts. This resulted in a major down-regulation of alpha7 protein expression. Promoter assays revealed that C2C12 myoblasts transfected with deltaEF1 showed a decrease in activity of the 2.8-kb alpha7 promoter fragment, indicating regulation of alpha7 integrin at the transcriptional level. We have identified two E-box-like sites for deltaEF1 in the negative regulatory region. Mutation of these sites enhanced alpha7 promoter activity, indicating that these sites function in repression. MYOD, an activator of alpha7 integrin transcription, can compete with deltaEF1 for binding at these sites in gel shift assay. By using chromatin immunoprecipitation, we demonstrated a reciprocal binding of deltaEF1 and MYOD to this regulatory element depending on the stage of differentiation: deltaEF1 is preferentially bound in myoblasts to this region, whereas MYOD is bound in myotubes. The N-terminal region of deltaEF1 is necessary for alpha7 repression, and this region also binds the co-activator p300/CBP. Importantly, we found that the p300/CBP co-activator can overcome repression by deltaEF1, suggesting that deltaEF1 can titrate limiting amounts of this co-activator. These findings suggest that deltaEF1 has a role in suppressing integrin expression in myoblasts by displacing MYOD and competing for p300/CBP co-activator.

Tumor cell invasion and survival in head and neck cancer.

Squamous cell carcinoma (SCC) is the primary tumor type in head and neck cancer. Typically, these tumor cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. The process of invasion involves concurrent infiltration and destruction of adjacent tissues. As with normal mucosal epithelium, SCC cells express receptors that mediate cell-extracellular matrix (ECM) adhesion (integrins) and cell-cell adhesion (cadherins). Both receptor families represent important signaling devices that are capable of promoting survival and proliferation. Recent results indicate that integrins and cadherins cooperate to regulate invasive behavior. During SCC invasion, cells actively migrate through the surrounding ECM with the simultaneous remodeling of their intercellular adhesions. During invasion, integrin receptor engagement with specific ECM ligands along with concurrent remodeling of cadherin adhesions induces changes in the cytoskeleton though modulation of the activities of Rho family members. Tumor development and progression of SCC proceeds with the generation of variant cells with potential alterations in expression of adhesion receptors, and their associated signaling pathways lead to a highly invasive and metastatic phenotype. Understanding the molecular events that define this subset of invasive cells will facilitate the development of new treatment strategies.

Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1.

Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the alpha3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the alpha2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-alpha2beta1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-alpha3beta1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.

Adhesion-mediated squamous cell carcinoma survival through ligand-independent activation of epidermal growth factor receptor.

The survival and growth of squamous epithelial cells require signals generated by integrin-matrix interactions. After conversion to squamous cell carcinoma, the cells remain sensitive to detachment-induced anoikis, yet in tumor cell aggregates, which are matrix-deficient, these cells are capable of suprabasal survival and proliferation. Their survival is enhanced through a process we call synoikis, whereby junctional adhesions between neighboring cells generate specific downstream survival signals. Here we show that in squamous cell carcinoma cells, E-cadherin-mediated cell-cell contacts specifically induce activation of epidermal growth factor receptor (EGFR). EGFR activation in turn triggers the ERK/MAPK signaling module, leading to elevation of anti-apoptotic Bcl-2. After intercellular adhesion, formation of adherens junctions triggers the formation of E-cadherin-EGFR complexes, correlating with EGFR transactivation. Analysis of the process with a dominant-negative EGFR mutant indicated that activation of EGFR is ligand-independent. Our data implicate cell-cell adhesion-induced activation of EGFR as a cooperative mechanism that generates compensatory survival signaling, protecting malignant cells from detachment-induced death.

Promoter methylation regulates cadherin switching in squamous cell carcinoma.

Cadherins are cell adhesion molecules that modulate the epithelial phenotype and regulate tumor invasion. To identify the role of promoter methylation in regulating E-cadherin expression and in the "switching" of cadherins in oral squamous cell carcinoma (SCC), we studied 14 cell lines for cadherin expression. Immunoblotting revealed that only two (HOC-313 and HA-376) showed strong up-regulation of N-cadherin, and neither expressed E-cadherin. These results were confirmed by PCR. Furthermore, analysis of genomic DNA showed that the lack of E-cadherin expression in the two cell lines was not due to gene deletion. In both cell lines, methylation-specific PCR indicated extensive methylation of the 5' CpG island in the E-cadherin promoter. After treatment with a DNA methylation inhibitor (5-Aza-2-deoxycytidine), both immunoblotting and immunofluorescence staining showed that HA-376 cells newly expressed E-cadherin with a parallel decrease in their N-cadherin expression. Multiplex RT-PCR demonstrated that the down-regulation of N-cadherin mRNA was coordinately regulated with E-cadherin expression. Thus, methylation of the 5' CpG island in the E-cadherin promoter induces reciprocal expression of E- and N-cadherins in oral SCC by an unknown mechanism that appears to be mediated at the level of N-cadherin gene expression. These events may play an important role in the regulation of tumor cell mobility and invasion.

Regulation of alpha7 integrin expression during muscle differentiation.

Expression of the laminin-binding alpha7 integrin is tightly regulated during myogenic differentiation, reflecting required functions that range from cell motility to formation of stable myotendinous junctions. However, the exact mechanism controlling alpha7 expression in a tissue- and differentiation-specific manner is poorly understood. This report provides evidence that alpha7 gene expression during muscle differentiation is regulated by the c-Myc transcription factor. In myoblasts, alpha7 is expressed at basal levels, but following conversion to myotubes the expression of the integrin is strongly elevated. The increased alpha7 mRNA and protein levels following myogenic differentiation are inversely correlated with c-Myc expression. Transfection of myoblasts with the c-Myc transcription factor down-regulated alpha7 expression, whereas overexpression of Madmyc, a dominant-negative c-Myc chimera, induced elevated alpha7 expression. Functional analysis with site-specific deletions identified a specific double E-box sequence in the upstream promoter region (-2.0 to -2.6 kb) that is responsible for c-Myc-induced suppression of alpha7 expression. DNA-protein binding assays and supershift analysis revealed that c-Myc forms a complex with this double E-box sequence. Our results suggest that the interaction of c-Myc with this promoter region is an important regulatory element controlling alpha7 integrin expression during muscle development and myotendinous junction formation.

The beta1 cytoplasmic domain regulates the laminin-binding specificity of the alpha7X1 integrin.

During muscle development, the laminin-specific alpha7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the alpha7X1 or the alpha7X2 variant. The relative level of alpha7X1 and alpha7X2 is developmentally regulated. Similarly, the partner beta1 integrin cytoplasmic domain is converted from the beta1A to the beta1D splice variant. To determine whether beta1D modulates the activity of the alpha7 receptor, cells were transfected with alpha7X1 and beta1D cDNA. alpha7X1 coupled with beta1A failed to adhere to laminin-1, whereas cotransfectants expressing alpha7X1 and beta1D showed strong adhesion. Interestingly, alpha7X1 complexed with beta1A and beta1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that alpha7 function is regulated not only by X1/X2 in its extracellular domain but also by beta1 cytoplasmic splice variants. It is likely that expression of beta1D alters alpha7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of alpha7beta1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.

The extracellular matrix protein betaIG-H3 is expressed at myotendinous junctions and supports muscle cell adhesion.

Molecules of the extracellular matrix (ECM) play important roles in the development and maintenance of myotendinous junctions (MTJs), specialized regions of muscle to bone union. In this report we provide evidence that skeletal muscle cells synthesize the collagen- and fibronectin-binding ECM protein betaIG-H3 and that betaIG-H3 is localized to MTJs. In situ hybridization experiments revealed that during E16.5-E18.5 of murine development, betaIG-H3 RNA transcripts were expressed where developing skeletal muscle fibers contact primordial cartilage and bone. Immunohistochemical analysis verified that the betaIG-H3 protein itself localized distinctively at MTJs, and ultrastructural analysis suggested that betaIG-H3 associates with extracellular fibers and the surface of cells. In vitro, recombinant betaIG-H3 functioned as an adhesion substratum for skeletal muscle cells. Adhesion was significantly reduced by anti-integrin alpha7 and beta1 antibodies, suggesting that betaIG-H3 binds to skeletal muscle cells via alpha7beta1 integrin. Localization of betaIG-H3 to the termini of skeletal muscle fibers and the binding of betaIG-H3 to cells and to molecules of the ECM suggests that betaIG-H3 may play an organizational and structural role in developing MTJs, linking skeletal muscle to components of the ECM.

Dominant-negative E-cadherin alters adhesion and reverses contact inhibition of growth in breast carcinoma cells.

Cadherins play a crucial role in epithelial morphogenesis and mediate intercellular adhesion. These receptors bind catenins and are involved in signal transduction pathways that regulate cell growth and apoptosis, and are frequently down-regulated in invasive and metastatic carcinomas. In order to assess the role of E-cadherin in cell adhesion and growth, we transfected MCF-7 cells, a human breast cancer cell line, with a dominant-negative construct of E-cadherin (H-2kd-E-cad). The dominant-negative form of E-cadherin disrupted cell-cell adhesion of monolayer cells and induced an epithelial-to-fibroblastic conversion without any significant change in integrin profiles. Whereas control cells rapidly formed multicellular aggregates that tightly compacted into spheroids, dominant-negative transfected cells failed to compact and remained as loosely-associated cells. The transfectants exhibited down-regulation and redistribution of endogenous E-cadherin as well as increased levels of alpha- and beta-catenin. Importantly, the H-2kd-E-cad-transfected cells, when grown as multicellular aggregates, showed an increase in cell proliferation rate, compared to control cells. Overall, these observations suggest that in breast carcinoma, disruption of E-cadherin and catenin function modulates both cell-cell adhesion and permits escape from cell-cell contact-involved inhibition of cell growth.

Oral mucosal carcinogenesis in SENCAR mice.

Animal models of oral carcinogenesis have been developed but most use the hamster buccal pouch or rat oral mucosa. With completion of human and murine genome sequencing, the development of a mouse model of oral carcinogenesis may prove useful for future genomic studies of oral carcinogenesis. To achieve this objective, 30 SENCAR mice were initiated by brush application of palatal, buccal and tongue mucosa with 200 nmol 7,12-dimethylbenz[a]anthracene (DMBA) using 3 treatment regimens, and promoted by brush application with 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) for a total of 28 weeks. Alternatively, 5 mice were treated with 0.5% 4-nitroquinoline-1-oxide (4NQO) alone by brush application for 28 weeks. There were another 6 control mice treated with vehicle alone. The tumor samples were analyzed for the presence of H-ras codon 61 gene mutations using a mutant-allele-specific amplification-polymerase chain reaction (MASA-PCR) technique. The results showed that among the group of 24 mice initiated with DMBA for 2 or 6 weeks, a range of papilliferous lesions were seen on the buccal mucosa comprising papillomas, papillomas with dysplasia and 7 squamous cell carcinomas (SCC). In those 6 mice initiated with 1 week of DMBA, only papillomas developed. In the 5 mice treated with 4NQO, one developed papillomas with dysplasia and two had SCCs in the tongue mucosa but not the buccal mucosa. Both carcinogens induced codon 61 mutation of the H-ras gene at a high frequency. The results indicated that DMBA/TPA and 4NQO in SENCAR mice reliably produced preneoplastic and malignant oral cavity lesions, which resemble the multistages for human oral carcinogenesis, and targeted to site-specific zones of the oral mucosa, namely the buccal mucosa and tongue, respectively. These results show that SENCAR mice can be used as a unique model of oral carcinogenesis with the potential for detailed molecular studies of neoplastic progression to SCC.