Douglas B. Spicer

Twist1 dimer selection regulates cranial suture patterning and fusion

Saethre‐Chotzen syndrome is associated with haploinsufficiency of the basic‐helix–loop–helix (bHLH) transcription factor TWIST1 and is characterized by premature closure of the cranial sutures, termed craniosynostosis; however, the mechanisms underlying this defect are unclear. Twist1 has been shown to play both positive and negative roles in mesenchymal specification and differentiation, and here we show that the activity of Twist1 is dependent on its dimer partner. Twist1 forms both homodimers (T/T) and heterodimers with E2A E proteins (T/E) and the relative level of Twist1 to the HLH inhibitor Id proteins determines which dimer forms. On the basis of the expression patterns of Twist1 and Id1 within the cranial sutures, we hypothesized that Twist1 forms homodimers in the osteogenic fronts and T/E heterodimers in the mid‐sutures. In support of this hypothesis, we have found that genes regulated by T/T homodimers, such as FGFR2 and periostin, are expressed in the osteogenic fronts, whereas genes regulated by T/E heterodimers, such as thrombospondin‐1, are expressed in the mid‐sutures. The ratio between these dimers is altered in the sutures of Twist1+/− mice, favoring an increase in homodimers and an expansion of the osteogenic fronts. Of interest, the T/T to T/E ratio is greater in the coronal versus the sagittal suture, and this finding may contribute to making the coronal suture more susceptible to fusion due to TWIST haploinsufficiency. Importantly, we were able to inhibit suture fusion in Twist1+/− mice by modulating the balance between these dimers toward T/E formation, by either increasing the expression of E2A E12 or by decreasing Id expression. Therefore, we have identified dimer partner selection as an important mediator of Twist1 function and provide a mechanistic understanding of craniosynostosis due to TWIST haploinsufficiency. Developmental Dynamics 235:1345–1357, 2006.

Twist1 homodimers enhance FGF responsiveness of the cranial sutures and promote suture closure

Haploinsufficiency of the transcription factor TWIST1 is associated with Saethre-Chotzen Syndrome and is manifested by craniosynostosis, which is the premature closure of the calvaria sutures. Previously, we found that Twist1 forms functional homodimers and heterodimers that have opposing activities. Our data supported a model that within the calvaria sutures Twist1 homodimers (T/T) reside in the osteogenic fronts while Twist1/E protein heterodimers (T/E) are in the mid-sutures. Twist1 haploinsufficiency alters the balance between these dimers, favoring an increase in homodimer formation throughout the sutures. The data we present here further supports this model and extends it to integrate the Twist1 dimers with the pathways that are known to regulate cranial suture patency. This data provides the first evidence of a functional link between Twist1 and the FGF pathway, and indicates that differential regulation of FGF signaling by T/T and T/E dimers plays a central role in governing cranial suture patency. Furthermore, we show that inhibition of FGF signaling prevents craniosynostosis in Twist1(+/-) mice, demonstrating that inhibition of a signaling pathway that is not part of the initiating mutation can prevent suture fusion in a relevant genetic model of craniosynostosis.

EMT Inducers Catalyze Malignant Transformation of Mammary Epithelial Cells and Drive Tumorigenesis towards Claudin-Low Tumors in Transgenic Mice

The epithelial-mesenchymal transition (EMT) is an embryonic transdifferentiation process consisting of conversion of polarized epithelial cells to motile mesenchymal ones. EMT–inducing transcription factors are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. Supporting oncogenic activity within primary lesions, the TWIST and ZEB proteins can prevent cells from undergoing oncogene-induced senescence and apoptosis by abolishing both p53- and RB-dependent pathways. Here we show that they also downregulate PP2A phosphatase activity and efficiently cooperate with an oncogenic version of H-RAS in malignant transformation of human mammary epithelial cells. Thus, by down-regulating crucial tumor suppressor functions, EMT inducers make cells particularly prone to malignant conversion. Importantly, by analyzing transformed cells generated in vitro and by characterizing novel transgenic mouse models, we further demonstrate that cooperation between an EMT inducer and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors, including low expression of tight and adherens junction genes, EMT traits, and stem cell–like characteristics. Claudin-low tumors are believed to be the most primitive breast malignancies, having arisen through transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Challenging this prevailing view, we propose that these aggressive tumors arise from cells committed to luminal differentiation, through a process driven by EMT inducers and combining malignant transformation and transdifferentiation.

Notch and Transforming Growth Factor-β (TGFβ) Signaling Pathways Cooperatively Regulate Vascular Smooth Muscle Cell Differentiation

Notch and transforming growth factor-β (TGFβ) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFβ1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle α-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFβ1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-α (SM22α) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFβ1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFβ1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFβ1 treatment led to synergistic activation of a TGFβ-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22α promoters. Thus, Notch and TGFβ coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

A Loss-of-Function Polymorphism in the Propeptide Domain of the LOX Gene and Breast Cancer

The lysyl oxidase (LOX) gene reverted Ras transformation of NIH 3T3 fibroblasts and tumor formation by gastric cancer cells, which frequently carry mutant RAS genes. The secreted lysyl oxidase proenzyme is processed to a propeptide (LOX-PP) and a functional enzyme (LOX). Unexpectedly, the tumor suppressor activity mapped to the LOX-PP domain, which inhibited tumor formation and the invasive phenotype of NF639 breast cancer cells driven by human epidermal growth factor receptor-2/neu, which signals via Ras. A single-nucleotide polymorphism, G473A (rs1800449), resulting in an Arg158Gln substitution in a highly conserved region within LOX-PP, occurs with an average 473A allele carrier frequency of 24.6% in the HapMap database, but was present in many breast cancer cell lines examined. Here, we show that the Arg-to-Gln substitution profoundly impairs the ability of LOX-PP to inhibit the invasive phenotype and tumor formation of NF639 cells in a xenograft model. LOX-PP Gln displayed attenuated ability to oppose the effects of LOX, which promoted a more invasive phenotype. In a case-control study of African American women, a potential association of the Gln-encoding A allele was seen with increased risk of estrogen receptor (ER)-alpha-negative invasive breast cancer in African American women. Consistently, LOX gene expression was higher in ER-negative versus ER-positive primary breast cancers, and LOX-PP Gln was unable to inhibit invasion by ER-negative cell lines. Thus, these findings identify for the first time genetic polymorphism as a mechanism of impaired tumor suppressor function of LOX-PP and suggest that it may play an etiologic role in ER-negative breast cancer.

Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves.

During embryogenesis the heart valves develop from undifferentiated mesenchymal endocardial cushions (EC), and activated interstitial cells of adult diseased valves share characteristics of embryonic valve progenitors. Twist1, a class II basic-helix-loop-helix (bHLH) transcription factor, is expressed during early EC development and is down-regulated later during valve remodeling. The requirements for Twist1 down-regulation in the remodeling valves and the consequences of prolonged Twist1 activity were examined in transgenic mice with persistent expression of Twist1 in developing and mature valves. Persistent Twist1 expression in the remodeling valves leads to increased valve cell proliferation, increased expression of Tbx20, and increased extracellular matrix (ECM) gene expression, characteristic of early valve progenitors. Among the ECM genes predominant in the EC, Col2a1 was identified as a direct transcriptional target of Twist1. Increased Twist1 expression also leads to dysregulation of fibrillar collagen and periostin expression, as well as enlarged hypercellular valve leaflets prior to birth. In human diseased aortic valves, increased Twist1 expression and cell proliferation are observed adjacent to nodules of calcification. Overall, these data implicate Twist1 as a critical regulator of valve development and suggest that Twist1 influences ECM production and cell proliferation during disease.

Two novel human NUMB isoforms provide a potential link between development and cancer.

We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

Conditional expression of Spry1 in neural crest causes craniofacial and cardiac defects

BackgroundGrowth factors and their receptors are mediators of organogenesis and must be tightly regulated in a temporal and spatial manner for proper tissue morphogenesis. Intracellular regulators of growth factor signaling pathways provide an additional level of control. Members of the Sprouty family negatively regulate receptor tyrosine kinase pathways in several developmental contexts. To gain insight into the role of Spry1 in neural crest development, we analyzed the developmental effects of conditional expression of Spry1 in neural crest-derived tissues.ResultsHere we report that conditional expression of Spry1 in neural crest cells causes defects in craniofacial and cardiac development in mice. Spry1;Wnt1-Cre embryos die perinatally and exhibit facial clefting, cleft palate, cardiac and cranial nerve defects. These defects appear to be the result of decreased proliferation and increased apoptosis of neural crest and neural crest-derived cell populations. In addition, the domains of expression of several key transcription factors important to normal craniofacial and cardiac development including AP2, Msx2, Dlx5, and Dlx6 were reduced in Spry1;Wnt1-Cre transgenic embryos.ConclusionCollectively, these data suggest that Spry1 is an important regulator of craniofacial and cardiac morphogenesis and perturbations in Spry1 levels may contribute to congenital disorders involving tissues of neural crest origin.

Twist1 Controls a Cell-Specification Switch Governing Cell Fate Decisions within the Cardiac Neural Crest

Neural crest cells are multipotent progenitor cells that can generate both ectodermal cell types, such as neurons, and mesodermal cell types, such as smooth muscle. The mechanisms controlling this cell fate choice are not known. The basic Helix-loop-Helix (bHLH) transcription factor Twist1 is expressed throughout the migratory and post-migratory cardiac neural crest. Twist1 ablation or mutation of the Twist-box causes differentiation of ectopic neuronal cells, which molecularly resemble sympathetic ganglia, in the cardiac outflow tract. Twist1 interacts with the pro-neural factor Sox10 via its Twist-box domain and binds to the Phox2b promoter to repress transcriptional activity. Mesodermal cardiac neural crest trans-differentiation into ectodermal sympathetic ganglia-like neurons is dependent upon Phox2b function. Ectopic Twist1 expression in neural crest precursors disrupts sympathetic neurogenesis. These data demonstrate that Twist1 functions in post-migratory neural crest cells to repress pro-neural factors and thereby regulate cell fate determination between ectodermal and mesodermal lineages.

Identification of responsive cells in the developing somite supports a role for β-catenin-dependent Wnt signaling in maintaining the DML myogenic progenitor pool

Somitic β‐catenin is involved in both maintaining a stem cell population and controlling myogenic differentiation. It is unclear how β‐catenin‐dependent Wnt signaling accomplishes these disparate roles. The present study shows that only dorsal cells in the early somite respond to β‐catenin‐dependent Wnt signaling and as the somites compartmentalize to form the dermomyotome and myotome, responding cells are detected primarily in the dorsomedial lip (DML). Forced activation of Wnt target genes in DML cells prevents their progeny from entering the myotome, while blocking activation allows myotomal entry. This suggests a role for β‐catenin‐dependent/Wnt signaling in maintaining progenitor cells in the DML and that if β‐catenin‐dependent/Wnt signaling is required to induce myogenesis, the response is transitory and rapidly down‐regulated. Developmental Dynamics 239:222–236, 2010.