M. Michele Pisano

Identification of three novel Smad binding proteins involved in cell polarity

A yeast two‐hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par‐3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S‐transferase) pull‐down assays, Erbin, Par‐3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full‐length Smad 3 protein. Failure of Erbin, Par‐3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par‐3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor β (TGFβ) has been suggested to alter cell polarity through a Smad‐independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFβ could alter cell polarity via a Smad‐dependent signaling mechanism.

Cross‐talk between the TGFβ and Wnt signaling pathways in murine embryonic maxillary mesenchymal cells

The transforming growth factor beta (TGFβ) and Wnt signaling pathways play central roles regulating embryogenesis and maintaining adult tissue homeostasis. TGFβ mediates its cellular effects through types I and II cell surface receptors coupled to the nucleocytoplasmic Smad proteins. Wnt signals via binding to a cell surface receptor, Frizzled, which in turn activates intracellular Dishevelled, ultimately leading to stabilization and nuclear translocation of β‐catenin. Previous studies have demonstrated several points of cross‐talk between the TGFβ and Wnt signaling pathways. In yeast two‐hybrid and GST‐pull down assays, Dishevelled‐1 and Smad 3 have been shown to physically interact through the C‐terminal one‐half of Dishevelled‐1 and the MH2 domain of Smad 3. The current study demonstrates that co‐treatment of murine embryonic maxillary mesenchyme (MEMM) cells with Wnt‐3a and TGFβ leads to enhanced reporter activity from TOPflash, a Wnt‐responsive reporter plasmid. Transcriptional cooperation between TGFβ and Wnt did not require the presence of a Smad binding element, nor did it occur when a TGFβ‐responsive reporter plasmid (p3TP‐lux) was transfected. Overexpression of Smad 3 further enhanced the cooperation between Wnt and TGFβ while overexpression of dominant‐negative Smads 2 and 3 inhibited this effect. Co‐stimulation with TGFβ led to greater nuclear translocation of β‐catenin, providing explanation for the effect of TGFβ on Wnt‐3a reporter activity. Wnt‐3a exerted antiproliferative activity in MEMM cells, similar to that exerted by TGFβ. In addition, Wnt‐3a and TGFβ in combination led to synergistic decreases in MEMM cell proliferation. These data demonstrate a functional interaction between the TGFβ and Wnt signaling pathways and suggest that Wnt activation of the canonical pathway is an important mediator of MEMM cell growth.

Intracellular dynamics of Smad-mediated TGFβ signaling

The transforming growth factor‐β (TGFβ) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFβ on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFβ action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFβ. In the current study, elements of the Smad component of the TGFβ intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFβ1, TGFβ2, and TGFβ3 was demonstrated. TGFβ‐initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFβ‐inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad‐mediated TGFβ signaling system in cells of the developing murine palate. J. Cell. Physiol. 197: 261–271, 2003.

CDC2-related kinase PITALRE phosphorylates pRb exclusively on serine and is widely expressed in human tissues

Mammalian cell cycle progression is regulated by sequential activation and inactivation of cyclin‐dependent kinases (cdks). Recently, several new members of the cdk family were cloned, and some of these were shown to complex with different cyclins and to be active at discrete stages of the cell cycle. PITALRE, a new member of this family, was cloned by our laboratory and was shown to be able to phosphorylate pRb protein in vitro. In the current work, we found that PITALRE kinase activity phosphorylated pRb at sites similar to those phosphorylated by the CDC2 kinase, which itself is known to mimic, in vitro, the in vivo phosphorylation of pRb. Phosphorylation of pRb by the PITALRE‐associated kinase activity was on Ser residues exclusively. Moreover, we investigated the expression pattern of PITALRE in normal human tissues, using immunohistochemical techniques so as to gain additional data on the characteristics of this new cdk family member. The protein was widely expressed, although a different tissue distribution and/or level of expression was found in various organs. Some specialized tissues such as blood, lymphoid tissue, ovarian cells, and the endocrine portion of the pancreas showed a high expression level of PITALRE. The specific expression pattern found suggests that PITALRE may be involved in specialized functions in certain cell types. J. Cell. Physiol. 172:265–273, 1997.

Alcohol modulates expression of DNA methyltranferases and methyl CpG-/CpG domain-binding proteins in murine embryonic fibroblasts.

Fetal alcohol syndrome (FAS), presenting with a constellation of neuro-/psychological, craniofacial and cardiac abnormalities, occurs frequently in offspring of women who consume alcohol during pregnancy, with a prevalence of 1-3 per 1000 livebirths. The present study was designed to test the hypothesis that alcohol alters global DNA methylation, and modulates expression of the DNA methyltransferases (DNMTs) and various methyl CpG-binding proteins. Murine embryonic fibroblasts (MEFs), utilized as an in vitro embryonic model system, demonstrated ∼5% reduction in global DNA methylation following exposure to 200mM ethanol. In addition, ethanol induced degradation of DNA methyltransferases (DNMT-1, DNMT-3a, and DNMT-3b), as well as the methyl CpG-binding proteins (MeCP-2, MBD-2 and MBD-3), in MEF cells by the proteasomal pathway. Such degradation could be completely rescued by pretreatment of MEF cells with the proteasomal inhibitor, MG-132. These data support a potential epigenetic molecular mechanism underlying the pathogenesis of FAS during mammalian development.

Developmental MicroRNA Expression Profiling of Murine Embryonic Orofacial Tissue

BACKGROUND Orofacial development is a multifaceted process involving precise, spatio-temporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs involved in gene silencing, represent critical regulators of cell and tissue differentiation. MicroRNA gene expression profiling is an effective means of acquiring novel and valuable information regarding the expression and regulation of genes, under the control of miRNA, involved in mammalian orofacial development. METHODS To identify differentially expressed miRNAs during mammalian orofacial ontogenesis, miRNA expression profiles from gestation day (GD) -12, -13 and -14 murine orofacial tissue were compared utilizing miRXplore microarrays from Miltenyi Biotech. Quantitative real-time PCR was utilized for validation of gene expression changes. Cluster analysis of the microarray data was conducted with the clValid R package and the UPGMA clustering method. Functional relationships between selected miRNAs were investigated using Ingenuity Pathway Analysis. RESULTS Expression of over 26% of the 588 murine miRNA genes examined was detected in murine orofacial tissues from GD-12-GD-14. Among these expressed genes, several clusters were seen to be developmentally regulated. Differential expression of miRNAs within such clusters wereshown to target genes encoding proteins involved in cell proliferation, cell adhesion, differentiation, apoptosis and epithelial-mesenchymal transformation, all processes critical for normal orofacial development. CONCLUSIONS Using miRNA microarray technology, unique gene expression signatures of hundreds of miRNAs in embryonic orofacial tissue were defined. Gene targeting and functional analysis revealed that the expression of numerous protein-encoding genes, crucial to normal orofacial ontogeny, may be regulated by specific miRNAs.

MicroRNA gene expression signatures in the developing neural tube.

BACKGROUND Neurulation requires precise, spatio-temporal expression of numerous genes and coordinated interaction of signal transduction and gene regulatory networks, disruption of which may contribute to the etiology of neural tube defects (NTDs). MicroRNAs (miRNAs) are key modulators of cell and tissue differentiation. To define potential roles of miRNAs in development of the murine neural tube (NT), miRNA microarray analysis was conducted to establish expression profiles, and identify miRNA target genes and functional gene networks. METHODS The miRNA expression profiles in murine embryonic NTs derived from gestational days 8.5, 9.0, and 9.5 were defined and compared utilizing miRXplore microarrays from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. Gene expression changes were verified by TaqMan quantitative Real-Time PCR. The clValid R package and the UPGMA (hierarchical) clustering method were utilized for cluster analysis of the microarray data. Functional associations among selected miRNAs were examined via Ingenuity Pathway Analysis. RESULTS The miRXplore chips enabled examination of 609 murine miRNAs. Expression of approximately 12% of these was detected in murine embryonic NTs. Clustering analysis revealed several developmentally regulated expression clusters among these expressed genes. Target analysis of differentially expressed miRNAs enabled identification of numerous target genes associated with cellular processes essential for normal NT development. Utilization of Ingenuity Pathway Analysis revealed interactive biologic networks which connected differentially expressed miRNAs with their target genes, and highlighted functional relationships. CONCLUSIONS The present study defined unique gene expression signatures of a range of miRNAs in the developing NT during the critical period of NT morphogenesis. Analysis of miRNA target genes and gene interaction pathways revealed that specific miRNAs might direct expression of numerous genes encoding proteins, which have been shown to be indispensable for normal neurulation. This study is the first to identify miRNA expression profiles and their potential regulatory networks in the developing mammalian NT.

Expression of the E2F and retinoblastoma families of proteins during neural differentiation

Development of the brain is determined by a strictly orchestrated program of proliferation, migration, apoptosis, differentiation, synaptogenesis, tract formation, and myelination. The E2F family of transcription factors, whose activity and functions are regulated in large part through interactions with the retinoblastoma (Rb) family of tumor suppressor proteins, has been implicated as a key regulator of proliferation, differentiation, and apoptosis in a variety of tissues. We have examined levels of the E2F and Rb families of proteins during both brain development and neural differentiation of P19 cells, and found the expression profiles during these two processes of neural development and maturation to be quite similar, i.e., strong up-regulation of p130, pronounced down-regulation of p107, moderate up-regulation of pRb, and significant down-regulation of most species of E2F and dimerization protein (DP). However, several specific isoforms, namely a 30 kDa form of DP-2, a 57 kDa species of E2F-3, a 59 kDa form of E2F-5 and the isoforms of E2F-1 recognized by the E2F-1 (KH-95) antibody were up-regulated suggesting that these particular isoforms of E2F and DP play a tissue-specific function in differentiation and maturation of nervous tissue. The potential role of the E2F/DP family of transcription factors in aspects of neural development and differentiation are considered.

CBP/p300 and associated transcriptional co-activators exhibit distinct expression patterns during murine craniofacial and neural tube development

Mutations in each of the transcriptional co-activator genes - CBP, p300, Cited2, Cart1 and Carm1 - result in neural tube defects in mice. The present study thus furnishes a complete and comparative temporal and spatial expression map of CBP/p300 and associated transcriptional co-activators, Cited2, Cart1 and Carm1 during the period of murine neural tube development (embryonic days 8.5 to 10.5). Each co-activator except Cart1 was expressed in the dorsal neural folds on E8.5. Although CBP and p300 are functionally interchangeable in vitro, their respective expression patterns diverge during embryogenesis before neural fold fusion is complete. CBP gene expression was lost from the neural folds by E8.75 and was thereafter weakly expressed in the maxillary region and limb buds, while p300 exhibited strong expression in the first branchial arch, limb bud and telencephalic regions on E9.5. Cart1 exhibited strong expression in the forebrain mesenchyme from E9.0 through E10.5. Although CBP, p300, Carm1 and Cited2 share temporal expression on E8.5, these co-activators have different spatial expression in mesenchyme and/or the neuroepithelium. Nevertheless, co-localization to the dorsal neural folds on E8.5 suggests a functional role in elevation and/or fusion of the neural folds. Target genes, and pathways that promote cranial neural tube fusion that are activated by CBP/p300/Carm1/Cited2/Cart1-containing transcriptional complexes await elucidation.

MmPalateMiRNA, an R package compendium illustrating analysis of miRNA microarray data

BackgroundMicroRNAs (miRNAs) constitute the largest family of noncoding RNAs involved in gene silencing and represent critical regulators of cell and tissue differentiation. Microarray expression profiling of miRNAs is an effective means of acquiring genome-level information of miRNA activation and inhibition, as well as the potential regulatory role that these genes play within a biological system. As with mRNA expression profiling arrays, miRNA microarrays come in a variety of platforms from numerous manufacturers, and there are a multitude of techniques available for reducing and analyzing these data.ResultsIn this paper, we present an analysis of a typical two-color miRNA microarray experiment using publicly available packages from R and Bioconductor, the open-source software project for the analysis of genomic data. Covered topics include visualization, normalization, quality checking, differential expression, cluster analysis, miRNA target identification, and gene set enrichment analysis. Many of these tools carry-over from the analysis of mRNA microarrays, but with some notable differences that require special attention. The paper is presented as a “compendium” which, along with the accompanying R package MmPalateMiRNA, contains all of the experimental data and source code to reproduce the analyses contained in the paper.ConclusionsThe compendium presented in this paper will provide investigators with an access point for applying the methods available in R and Bioconductor for analysis of their own miRNA array data.