Investigating Reports of Complex Regional Pain Syndrome: An Analysis of HPV-16/18-Adjuvanted Vaccine Post-Licensure Data.

Complex regional pain syndrome (CRPS) is a chronic pain disorder that typically follows trauma or surgery. Suspected CRPS reported after vaccination with human papillomavirus (HPV) vaccines led to temporary suspension of proactive recommendation of HPV vaccination in Japan. We investigated the potential CRPS signal in relation to HPV-16/18-adjuvanted vaccine (Cervarix®) by database review of CRPS cases with independent expert confirmation; a disproportionality analysis and analyses of temporality; an observed versus expected analysis using published background incidence rates; systematic reviews of aggregate safety data, and a literature review. The analysis included 17 case reports of CRPS: 10 from Japan (0.14/100,000 doses distributed) and seven from the United Kingdom (0.08/100,000). Five cases were considered by independent experts to be confirmed CRPS. Quantitative analyses did not suggest an association between CRPS and HPV-16/18-adjuvanted vaccine. Observed CRPS incidence after HPV-16/18 vaccination was statistically significantly below expected rates. Systematic database reviews using search terms varying in specificity and sensitivity did not identify new cases. No CRPS was reported during clinical development and no unexpected results found in the literature. There is not sufficient evidence to suggest an increased risk of developing CRPS following vaccination with HPV-16/18-adjuvanted vaccine. Post-licensure safety surveillance confirms the acceptable benefit-risk of HPV-16/18 vaccination.

Smad3 is a key nonredundant mediator of transforming growth factor beta signaling in Nme mouse mammary epithelial cells.

Smad2 and Smad3 are intracellular mediators of transforming growth factor beta (TGFbeta) signaling that share various biochemical properties, but data emerging from functional analyses in several cell types indicate that these two Smad proteins may convey distinct cellular responses. Therefore, we have investigated the individual roles of Smad2 and Smad3 in mediating the cytostatic and proapoptotic effects of TGFbeta as well as their function in epithelial-to-mesenchymal transition. For this purpose, we transiently depleted mouse mammary epithelial cells (Nme) of Smad2 and/or Smad3 mainly by a strategy relying on RNaseH-induced degradation of mRNA. The effect of such depletion on hallmark events of TGFbeta-driven epithelial-to-mesenchymal transition was analyzed, including dissolution of epithelial junctions, formation of stress fibers and focal adhesions, activation of metalloproteinases, and transcriptional regulation of acknowledged target genes. Furthermore, we investigated the effect of Smad2 and Smad3 knockdown on the TGFbeta-regulated transcriptome by microarray analysis. Our results identify Smad3 as a key factor to trigger TGFbeta-regulated events and ascribe tumor suppressor as well as oncogenic activities to this protein.

Sesn1 is a novel gene for left-right asymmetry and mediating nodal signaling.

Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling.

Involvement of Ets-1 transcription factor in inducing matrix metalloproteinase-2 expression by epithelial-mesenchymal transition in human squamous carcinoma cells.

Epithelial-mesenchymal transition (EMT) is a crucial event in cancer progression. We previously reported that EMT up-regulates matrix metalloproteinase-2 (MMP-2) expression in squamous cell carcinoma (SCC) cells. In this study, we showed that Tet Off-induced expression of Snail or SIP1, and treatment with TGF-beta1 induced EMT in terms of down-regulation of E-cadherin, and up-regulation of vimentin and MMP-2 expression with morphological changes. In SCC cells, SIP1 expression was induced by Snail and TGF-beta1, but Snail expression was not induced by SIP1 or TGF-beta1. However, expression of Snail but not SIP1 was strongly increased by TGF-beta1 in highly invasive SCC cells with mesenchymal phenotypes. Analysis of the MMP-2 promoter revealed that an Ets-1 binding site, located between position -1255 and -1248 relative to the transcriptional start site, was critical for the activation by Snail, SIP1 and TGF-beta1 in SCC cells. Induced expression of Snail and SIP1 resulted in the increased expression of Ets-1 and DNA-binding activities of nuclear proteins to the Ets-1-binding site and strong Ets-1 expression was detected in highly invasive SCC cells. Furthermore, overexpression of Ets-1 induced the promoter-activation and expression of MMP-2 without EMT. These results indicate that EMT induces Ets-1 expression, which activates the MMP-2 promoter, but Ets-1 by itself has no activity to induce EMT in SCC cells.

The novel Smad-interacting protein Smicl regulates Chordin expression in the Xenopus embryo.

In this paper, we investigate the function of Smicl, a zinc-finger Smad-interacting protein that is expressed maternally in the Xenopus embryo. Inhibition of Smicl function by means of antisense morpholino oligonucleotides causes the specific downregulation of Chordin, a dorsally expressed gene encoding a secreted BMP inhibitor that is involved in mesodermal patterning and neural induction. Chordin is activated by Nodal-related signalling in an indirect manner, and we show here that Smicl is involved in a two-step process that is necessary for this activation. In the first, Smad3 (but not Smad2) activates expression of Xlim1 in a direct fashion. In the second, a complex containing Smicl and the newly induced Xlim1 induces expression of Chordin. As well as revealing the function of Smicl in the early embryo, our work yields important new insight in the regulation of Chordin and identifies functional differences between the activities of Smad2 and Smad3 in the Xenopus embryo.

Smicl is a novel Smad interacting protein and cleavage and polyadenylation specificity factor associated protein.

Ligand-bound receptors of the Transforming Growth Factor-beta (TGF-beta) family promote the formation of complexes between Smad proteins that subsequently accumulate in the nucleus and interact there with other transcriptional regulators, leading to modulation of target gene expression. We identified a novel nuclear protein, Smicl, which binds to Smad proteins. Smicl and Smads cooperate and enhance TGF-beta mediated activation of a Smad-responsive reporter gene. A domain with five CCCH-type zinc fingers in Smicl is structurally and functionally, at least in vitro, similar to a domain in CPSF-30, the 30 kDa subunit of Cleavage and Polyadenylation Specificity Factor (CPSF). Like CPSF-30, Smicl can associate with some other CPSF subunits characterized previously. Its effect on the induction of a reporter gene for TGF-beta requires the cleavage/polyadenylation signal downstream of the coding sequence of that gene. Thus, Smicl is a novel protein that displays CPSF-30-like activities, interacts in the nucleus with activated Smads, and potentiates in TGF-beta stimulated cells Smad-dependent transcriptional responses, possibly in conjunction with the activity of CPSF complexes.

Smads and chromatin modulation.

Smad proteins are critical intracellular effector proteins and regulators of transforming growth factor type beta (TGFbeta) modulated gene transcription. They directly convey signals that initiate at ligand-bound receptor complexes and end in the nucleus with changes in programs of gene expression. Activated Smad proteins seem to recruit chromatin modifying proteins to target genes besides cooperating with DNA-bound transcription factors. We survey here the current and still emerging knowledge on Smad-binding factors, and their different mechanisms of chromatin modification in particular, in Smad-dependent TGFbeta signaling.

Transforming growth factor-beta-activated kinase-1 (TAK1), a MAP3K, interacts with Smad proteins and interferes with osteogenesis in murine mesenchymal progenitors.

TAK1 (transforming growth factor-beta-activated kinase-1), a MAP3K with considerable sequence similarity to Raf-1 and MEKK-1, has been identified as a transforming growth factor-beta/bone morphogenetic protein (BMP)-activated cytosolic component of the MAPK pathways. In this investigation, the molecular interactions between TAK1 and Smad proteins were characterized as well as their influence on BMP-mediated mesenchymal cell differentiation along the osteogenic/chondrogenic pathway. In co-immunoprecipitations we found an interaction of TAK1 with all Smads tested, R-Smads Smads1-5, the co-Smad Smad4, and the inhibitory Smads (I-Smad6 and I-Smad7). Smad interaction with TAK1 takes place through their MH2 domain. This interaction is dependent on the presence of an active kinase domain in TAK1. TAK1 dramatically interferes with R-Smad transactivation in reporter assays and affects subcellular distribution of Smad proteins. Activated TAK1 also interferes with BMP-dependent osteogenic development in murine mesenchymal progenitor cells (C3H10T 1/2). A potential TAK1-mediated apoptosis process could be excluded for these cells. Both synergistic and interfering influences of TAK1 on BMP-mediated Smad-signaling have been reported previously. We suggest that TAK1 is a factor that is involved in the fine-tuning of BMP effects during osteogenic development.

Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis.

Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.

Organization of the mouse Zfhx1b gene encoding the two-handed zinc finger repressor Smad-interacting protein-1.

SIP1, a member of the deltaEF1 family of two-handed zinc finger transcriptional repressors, has been identified as a Smad-binding protein. Recently, mutations in the human SIP1 gene (ZFHX1B) have been implicated in Hirschsprung disease. Here we document extensively the structure and transcriptional pattern of the mouse SIP1 gene (Zfhx1b) and compare it to homologues from other species. The overall structure of Zfhx1b is highly similar to that of the deltaEF1 gene (Zfhx1a), confirming their close evolutionary relationship. In contrast to Zfhx1a, the 5' untranslated region of the SIP1-encoding mouse gene is very complex and includes several alternative exons. The corresponding 5'-UTR splicing pattern seems to be conserved between species and suggests a role in its transcriptional and/or translational regulation. The gene also codes for an antisense transcript that is highly conserved between human and mouse.

Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells.

We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.

New intracellular components of bone morphogenetic protein/Smad signaling cascades.

Bone morphogenetic proteins (BMPs) regulate many processes in the embryo, including cell type specification, patterning, apoptosis, and epithelial-mesenchymal interaction. They also act in soft and hard tissues in adult life. Their signals are transduced from the plasma membrane to the nucleus through a limited number of Smad proteins. The list of Smad-interacting proteins is however growing and it is clear that these partners determine the outcome of the signal. We summarize the present status in BMP/Smad signaling, with emphasis on recently identified Smad partners and how these proteins may cooperate in the regulation of the expression of BMP target genes.

Interaction between Smad-interacting protein-1 and the corepressor C-terminal binding protein is dispensable for transcriptional repression of E-cadherin.

deltaEF1 and SIP1 (or Zfhx1a and Zfhx1b, respectively) are the only known members of the vertebrate Zfh1 family of homeodomain/zinc finger-containing proteins. Similar to other transcription factors, both Smad-interacting protein-1 (SIP1) and deltaEF1 are capable of repressing E-cadherin transcription through binding to the E2 boxes located in its promoter. In the case of deltaEF1, this repression has been proposed to occur via interaction with the corepressor C-terminal binding protein (CtBP). In this study, we show by coimmunoprecipitation that SIP1 and CtBP interact in vivo and that an isolated CtBP-binding SIP1 fragment depends on CtBP for transcriptional repression. However, and most importantly, full-length SIP1 and deltaEF1 proteins do not depend on their interaction with CtBP to repress transcription from the E-cadherin promoter. Furthermore, in E-cadherin-positive kidney epithelial cells, the conditional synthesis of mutant SIP1 that cannot bind to CtBP abrogates endogenous E-cadherin expression in a similar way as wild-type SIP1. Our results indicate that full-length SIP1 can repress E-cadherin in a CtBP-independent manner.

Complex Smad-dependent transcriptional responses in vertebrate development and human disease.

Smad proteins mediate genomic responses to polypeptides of the transforming growth factor type beta (TGF-beta) family, affecting cellular proliferation and differentiation, adhesion, and death. Members of one class of these Smad proteins, the receptor-regulated Smads or R-Smads, accumulate in the nucleus on their activation by ligand-bound complexes of serine-threonine kinase receptors at the cell surface. These effector proteins then participate directly in the regulation of gene expression by binding to DNA, interacting with transcription factors, and recruiting corepressors or coactivators to specific promoters. Although many nuclear Smad-interacting factors were isolated during the last 3 years, the field has recently taken a step beyond the characterization of the activity of these Smad-containing complexes on gene expression in vitro, as it addresses now their contribution to many processes in vivo. We have selected examples of such recent progress to illustrate the remarkable variation in the molecular mechanisms underlying Smad-dependent signaling depending on the non-Smad partner in the nucleus and their relevance to normal embryogenesis and consequences of their deregulation in human disorders and pathology.

Dynamic regulation of Brachyury expression in the amphibian embryo by XSIP1.

Xenopus Brachyury (Xbra) plays a key role in mesoderm formation during early development. One factor thought to be involved in the regulation of Xbra is XSIP1, a zinc finger/homeodomain-like DNA-binding protein that belongs to the deltaEF1 family of transcriptional repressors. We show here that Xbra and XSIP1 are co-expressed at the onset of gastrulation, but that expression subsequently refines such that Xbra is expressed in prospective mesoderm and XSIP1 in anterior neurectoderm. This refinement of the expression patterns of the two genes is due in part to the ability of XSIP1 to repress expression of Xbra. This repression is highly specific, in the sense that XSIP1 does not repress the expression of other regionally expressed genes in the early embryo, and that other members of the family to which XSIP1 belongs, such as deltaEF1 and its Xenopus homologue ZEB, cannot regulate Xbra expression. The function of XSIP1 was studied further by making an interfering construct comprising the open reading frame of XSIP1 fused to the VP16 transactivation domain. Experiments using this chimeric protein suggest that XSIP1 is required for normal gastrulation movements to occur and for the development of the anterior neural plate.