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M M Pisano

Publications and source records attributed to M M Pisano.

At least 19 recordsLinked to original sources
PubMed2003

Nuclear convergence of the TGFbeta and cAMP signal transduction pathways in murine embryonic palate mesenchymal cells.

Transforming growth factors beta (TGFbeta) and cyclic AMP (cAMP) both participate in growth and differentiation of the developing mammalian secondary palate and elicit similar biological responses. Cross-talk between these two signal transduction pathways in cells derived from the embryonic palate has been demonstrated previously. In the present study, we have examined nuclear convergence of these signalling pathways at the level of transcriptional complex formation. Biotinylated oligonucleotides encoding a consensus Smad binding element (SBE), or a cyclic AMP response element (CRE), were mixed with cell extracts from murine embryonic palate mesenchymal (MEPM) cells that were treated with either TGFbeta or forskolin. Protein-oligonucleotide complexes were precipitated with streptavidin-agarose, and analysed by Western blotting to identify proteins in the complex bound to each consensus oligonucleotide. TGFbeta treatment of MEPM cells increased the levels of phosphorylated Smad2, phosphorylated cAMP response element binding protein (CREB), and the coactivator, CREB binding protein (CBP), that were part of a complex bound to the SBE. Treatment of cells with forskolin, a stimulator of adenylate cyclase, increased the amount of phosphorylated CREB and CBP, but not the amount of phosphorylated Smad2 bound in a complex to the SBE. Additionally, the presence of the co-repressors, c-Ski and SnoN, was demonstrated as part of a complex bound to the SBE (but not the CRE). Amounts of c-Ski and SnoN found in the SBE-containing complex increased in response to either TGFbeta or forskolin. These results demonstrate that phosphorylated CREB forms a complex with the co-activator CBP, phosphorylated Smad2 and the co-repressors c-Ski and SnoN on a consensus SBE. This suggests cooperative regulation of genes with SBE-containing promoters by the cAMP and TGFbeta signalling pathways in the developing palate.

Animals↗
PubMed2003

Expression of the retinoblastoma family of tumor suppressors during murine embryonic orofacial development.

OBJECTIVE: To elucidate the role of the retinoblastoma (Rb) family of tumor suppressors and growth regulators in transforming growth factor beta (TGFbeta)-mediated embryonic palatal growth and morphogenesis. DESIGN: The spatio-temporal expression patterns of the RB1, RB2/p130, and p107 tumor suppressor genes, their gene products (pRb, p130 and p107) and phosphoforms were examined in the developing murine secondary palate utilizing reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot/immunolocalization analyses with phospho-specific antibodies. RESULTS: The RB1, RB2/p130, and p107 tumor suppressor genes and their gene products (pRb, p130, and p107) were differentially expressed in embryonic palatal tissue during the critical period of secondary palate development [gestational days (GD) 12-14]. Both hyper- (115 kDa) and hypo-phosphorylated (110 kDa) forms of pRb were expressed, with a notable transient decrease in expression on GD 13. Functional (hypo-phosphorylated) forms of pRB predominated during the critical period of palatogenesis. As opposed to pRb expression, p130 expression was transiently elevated on GD 13 in the embryonic palate, and functional (hypo-phosphorylated) forms were expressed at exceedingly low levels. p107 levels gradually declined over the course of palatogenesis. This diminution in expression, however, was accompanied by a transition to more functional (hypo-phosphorylated) forms of the p107 protein. When compared with Rb expression patterns in the whole embryo/fetus, each of the three Rb proteins exhibited unique and specific temporal patterns of expression in the developing palate. RT-PCR analyses of RB1, RB2/p130, and p107 mRNA expression in embryonic palatal tissue revealed patterns of expression which paralleled steady-state protein levels of pRb, p130, and p107 in the developing tissue. Immunolocalization of the Rb proteins demonstrated ubiquitous expression of pRb, p130, and p107 in embryonic palate mesenchyme and epithelium during GD 12-14 of development with intense nuclear staining of the Rbs in palate epithelial cells on days 12 and 13 of gestation. CONCLUSION: Changing patterns of pRb, p130, and p107 protein expression and phosphorylation were evident in the embryonic secondary palate during the course of palatal ontogenesis. As the function of all three Rb proteins is regulated primarily through their phosphorylation, this suggests significant variation in Rb protein functionality during the course of palate development. Additional evidence from our laboratory (manuscript in preparation) indicates that the TGFbetas, key regulators of palatal growth and morphogenesis, are able to differentially modulate phosphorylation of the Rb proteins in this developing tissue, and hence the TGFbetas may regulate the function(s) of the Rb proteins during palatal ontogenesis. Although functionality of the Rb proteins is known to be regulated primarily through post-translational modification (i.e. phopsphorylation), comparison of RB1, RB2/p130, and p107 steady-state mRNA levels with pRb, p130, and p107 steady-state protein levels in the developing palate levels suggests a significant degree of regulation at the transcriptional level. Differential patterns of Rb expression and phosphorylation in the developing palate suggest that pRb, p107, and p130 may each play unique roles in various aspects of growth, morphogenesis and cellular differentiation during palatal ontogenesis. Studies directed at elucidating the precise cellular role(s) of pRb, p107, and p130 during palate development, and at TGFbeta regulation of Rb expression/phosphorylation are ongoing in our laboratory.

Animals↗
PubMed2002

Expression of the nuclear coactivators CBP and p300 in developing craniofacial tissue.

cAMP regulatory element-binding protein (CREB)-binding protein (CBP) and its functional homolog, the adenovirus E1A -associated 300-kDa protein (p300) are nuclear coactivators and histone acetyltransferases that integrate signals from disparate pathways by bridging specific transcription factors to the basal transcription apparatus. Their role in patterning and development was suggested by studies in mice in which CBP and p300 expression was disrupted and by the human Rubinstein-Taybi syndrome, which is associated with mutations of CBP. The cAMP signal transduction pathway plays a critical role during development of the palate. The linkage between cAMP and expression of specific genes is mediated via activation of trans-acting deoxyribonucleic acid-binding proteins such as the nuclear CREB. For genes regulated by CBP- or p300-containing transcriptional complexes, rates of transcription will depend in part on cellular levels and distribution of CBP/p300. We have thus determined the temporal and spatial expression of CBP and p300 in murine embryonic palatal tissue. Both CBP and p300 proteins and messenger ribonucleic acids are expressed in palatal tissue on each d of palate development (days 12-14 of gestation), as measured by Western blotting and reverse-transcription polymerase chain reaction. Expression of both CBP and p300 was greatest on day 12 of gestation, suggesting that these transcriptional coactivators are developmentally regulated. Immunohistochemical analysis of CBP and p300 expression in the murine embryonic craniofacial region revealed a ubiquitous distribution for both proteins. These studies lay the groundwork for further investigations into the role of CBP and p300 in cellular signaling during craniofacial development.

Animals↗
PubMed2001

Convergence of cAMP, TGF-beta and retinoic acid signaling pathways in cells of the embryonic palate.

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-beta) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-beta1, cyclic adenosine 3', 5'-monophosphate (cAMP) and RA signaling systems. TGF-beta1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-beta mRNA in MEPM cells, though only TGF-beta1 inhibited RA-induced RAR-beta protein expression. Forskolin, but not TGF-beta1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-beta gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-beta-responsive, element. Thus, a putative TGF-beta Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-beta promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.

Animals↗
PubMed2001

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.

Animals↗
PubMed2000

Expression of the E2F family of transcription factors during murine development.

The E2F family of transcription factors plays a crucial role in the control of cell cycle progression and regulation of cellular proliferation, both processes fundamental to mammalian development. In the present study, we have examined the levels of expression of the six currently identified E2F proteins in murine embryos/fetuses as a function of gestational age, compared the expression of these six proteins in selected developing and adult tissues, and examined E2F expression in the embryonic murine palate, a tissue in which perturbation of proliferation is associated with induction of cleft palate. Our results indicate that: 1) multiple forms of individual E2F family members are present in embryonic, fetal and adult cells/tissues; 2) each of the six E2Fs is expressed in a tissue specific manner in both adult and embryonic/fetal organs; 3) certain forms of individual E2F family members are preferentially detected in adult tissues, whereas others are preferentially expressed in embryonic/fetal tissues; 4) expression of the various E2Fs and their isoforms follows distinct temporal patterns during murine gestation; and 5) individual E2F family members also exhibit differential patterns of temporal expression during murine palatogenesis.

Animals↗
PubMed1999

The role of RXR-alpha in retinoic acid-induced cleft palate as assessed with the RXR-alpha knockout mouse.

Treatment of pregnant mice with retinoic acid (RA) in mid-gestation produces cleft palate and limb defects in the fetuses. RXR-alpha has been previously shown to mediate the teratogenic effects of RA in the limb. In this study, we show that RXR-alpha is also involved in retinoid-induced palatal clefting. Treatment of RXR-alpha knockout mice with a teratogenic dose of RA on gestation day 11 or 12 induces cleft palate at a lower frequency than that seen in wild-type animals.

Animals↗
PubMed1998

Cloning of murine CDK9/PITALRE and its tissue-specific expression in development.

The cdc2-family of serine/threonine kinases and their binding partners recently were implicated in developmental roles. We previously cloned a cdc2-related kinase, cdk9/PITALRE, that is able to phosphorylate the retinoblastoma protein in vitro. We describe here the cloning and the characterization of the mouse homolog of cdk9/PITALRE. The murine cDNA is 98% identical with humans and is expressed at high levels in brain and kidney tissues. The kinase activity and protein expression of cdk9/PITALRE were highest in terminally differentiated tissues such as the muscle and brain. In situ immunohistology and immunofluorescence detected cdk9/PITALRE protein not only within terminally differentiated cells such as muscle and neuronal cells, but also in proliferating cells. C2C12 and P19 cells induced to differentiate along muscle and neural lineages peaked in cdk9/PITALRE kinase activity at the end of differentiation. These results suggest that, among other roles, cdk9/PITALRE plays a role not unlike cdk5 in the differentiation of certain cell types.

Amino Acid Sequence↗
PubMed1997

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.

Antibodies↗
PubMed1996

Teratogen-induced eye defects mediated by p53-dependent apoptosis.

BACKGROUND: Many birth defects are believed to involve gene-environment interactions, although the mechanisms involved are poorly understood. Apoptosis is a common effect of many kinds of environmental stresses on the developing embryo; therefore, mechanisms of teratogenesis may be approached within the context of the cell death program. The p53 tumor suppressor gene encodes a transcription factor which functions as a critical regulator of apoptosis in response to environmental stress. RESULTS: To investigate the relationship between p53-dependent apoptosis and teratogenesis, we subjected day 8 mouse embryos with different p53 gene backgrounds to a genotoxic stress, 2-chloro-2'-deoxyadenosine. Treatment rapidly stimulated nuclear p53 accumulation and triggered apoptosis in some (head-fold) but not other (primitive heart) developing structures. Induced cell death was p53 gene-dose dependent, as shown by the intermediate sensitivity of 4-5 somite stage embryos bearing only a single effective p53 allele and the lack of sensitivity of p53-null mutants. Abnormal development was manifested as eye defects by day 11, particularly lens agenesis. Overall the incidences of these defects at term were 73.3% for p53 wild-type fetuses, 52.5% for heterozygous mutants, and 2.2% for p53-null mutants. Statistical analysis indicated that the interaction between teratogen and genotype was highly significant (P < or = 0.001) for cell death on day 8 and eye defects on day 17. CONCLUSIONS: We conclude that teratogen induction of p53-dependent apoptosis in the developing embryo is positively coupled to the determination of congenital eye defects.

Abnormalities, Drug-Induced↗
PubMed1995

Isolation and characterization of the 5'-flanking sequence of the human ocular lens MIP gene.

The MIP (major intrinsic protein) gene, a member of an ancient family of membrane channel genes, encodes the predominant fiber cell membrane protein of the ocular lens. Its specific expression in the lens fibers is temporally and spatially regulated during development. To study the regulation of expression of MIP and delineate the regulatory elements underlying its tissue specificity and ontogenic profile, we have cloned 2840 bp of the human MIP 5'-flanking sequence. The human MIP 5'-flanking sequence contains three complete Alu repetitive elements in tandem at position between nt -1699 and -2684 (nt -1699/-2684). These Alu elements appear to have had a complex evolutionary history with insertions at different times. We have fused DNA fragments containing MIP 5'-flanking sequences to the bacterial cat reporter gene encoding chloramphenicol acetyltransferase and assayed them in primary cultures of chicken lens cells. We have mapped two negative regulatory regions in the human MIP 5'-flanking sequences -1564/-1696 and -948/-1000. We demonstrated that the human MIP 5'-flanking sequence -253/+42 contains a functional promoter in lens cells but is inactive in kidney epithelial cells or mouse fibroblasts, suggesting that this sequence contains regulatory elements responsible for the lens-specific expression of MIP.

Animals↗
PubMed1995

Patterns of cyclic AMP-dependent protein kinase gene expression during ontogeny of the murine palate.

Normal growth and differentiation of embryonic palatal tissue depends on regulated levels of intracellular cAMP. Cyclic AMP-dependent protein kinases (PKA) act to mediate the biological activities of cAMP. PKA isozyme protein profiles demonstrate a clear pattern of temporal alterations in embryonic palatal tissue during its development. In order to ascertain the molecular basis for changing PKA isozyme profiles during palatal ontogeny, the spatial and temporal expression of mRNAs for regulatory (RI alpha, RII alpha, and RII beta) and catalytic (C alpha) subunits of PKA was examined. RNA extracted from murine embryonic palatal tissue (days 12-14 of gestation) was examined by Northern blot analysis. Significant levels of constitutively expressed RI alpha and C alpha mRNA were seen on all days of gestation examined. RI alpha transcripts were substantially less abundant in palate mesenchymal cells in vitro than in palatal tissue in vivo. Levels of RII alpha and RII beta mRNA were highest on gestational day (GD) 12, a period characterized by pronounced palatal tissue growth. In addition, patterns of tissue distribution of RII beta, not previously described, were examined in the developing embryonic palate. A dramatic developmental shift in tissue distribution of RII beta was seen. The isozyme was evenly distributed between palatal epithelial and mesenchymal cells on GD 12 but by GD 14, RII beta was predominantly localized to palatal epithelial cells. Direct activation of adenylate cyclase with forskolin in murine embryonic palate mesenchymal (MEPM) cells resulted in an increase in RII alpha mRNA levels but had no effect on steady state levels of RII beta or C alpha mRNA. In addition, elevation of intracellular levels of cAMP resulted in a shift in the transcriptional profile of RI alpha mRNAs. Results of this study document specific patterns of expression for the genes encoding the various cAMP-dependent protein kinase regulatory and C alpha subunits in murine embryonic palatal tissue. In addition, we have demonstrated adaptational changes of this kinase in MEPM cells in response to conditions of increased intracellular levels of cAMP.

Animals↗
PubMed1995

Molecular cloning, expression, and developmental characterization of the murine retinoblastoma-related gene Rb2/p130.

The product of the retinoblastoma-related human gene Rb2/p130 is highly homologous with the product of the retinoblastoma tumor suppressor gene (pRb) and Rb-related p107. this homology is shared mainly in the pocket domain, a region that seems to play a key role in the functions of these proteins. Here we report the molecular cloning and initial characterization of the cDNA encoding the murine homologue of the human Rb2/p130 gene product. The 4.8-kb cDNA encodes a protein of 1125 amino acids that shows 90% indentity to that of the human protein. The Rb2/p130 mRNA is found to be expressed in all of the adult mouse tissues examined, with the highest level being detected in kidney and skeletal muscle. For the protein characterization, we used a polyclonal antibody raised against the COOH terminus of the human Rb2/p130 protein that also recognizes the mouse protein. In developing mouse embryos, the Rb2/p130 protein is expressed as early as day 10 of gestation and reached a peak of expression around day 13 of gestation, implying a developmental regulation of the Rb2/p130 gene in murine ontogeny.

3T3 Cells↗
PubMed1994

Regulation of TGF beta 3 gene expression in embryonic palatal tissue.

The TGF beta family of genes has been shown to play an important role in regulating various aspects of development, although the mechanisms by which TGF beta exerts its effects have not yet been clarified. Growth and differentiation of both murine embryonic palate mesenchymal (MEPM) cells and palatal epithelium can be regulated by the TGF beta s. We therefore examined the expression of mRNAs encoding TGF beta 1, TGF beta 2, and TGF beta 3 in developing embryonic palatal tissue as well as factors that modulate their levels of expression. Northern blot analysis of RNA isolated from murine embryonic palatal tissue on gestational days (GD) 12, 13, and 14 demonstrated the presence of one mRNA transcript for TGF beta 1 (2.5 kb), two transcripts for TGF beta 2 (4.4 kb, 6.0 kb), and one transcript for TGF beta 3 (3.5 kb). Although steady-state levels of TGF beta 1 mRNA showed no changes during development of the palate, TGF beta 2 mRNA levels were maximal on both GD13 and GD14 and TGF beta 3 mRNA levels transiently increased on GD 13. In addition, levels of TGF beta 3 mRNA seemed much higher than either TGF beta 1 or TGF beta 2. both TGF beta 1 and TGF beta 2 were able to increase, in a dose-related manner, the expression of TGF beta 3 mRNA in murine embryonic palate mesenchymal cells in vitro. In contrast, epidermal growth factor (EGF) down-regulated the expression of TGF beta 3 mRNA even in the presence of TGF beta 1 or TGF beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Ethanol effects on embryonic craniofacial growth and development: implications for study of the fetal alcohol syndrome.

Fetal alcohol syndrome (FAS), which is brought about by maternal consumption of ethanol during pregnancy, is a major public health problem. To gain understanding of the etiology of this condition, a number of teratological studies have been performed in different animal systems to develop an animal model for FAS. The C57BL/6J mouse strain has been described as susceptible to the teratogenic effects of ethanol, whereas the ICR (CD-1) strain is considered relatively insensitive. We have compared the effects of ethanol on DNA and protein synthesis in cultured embryonic palate mesenchymal cells from both strains to determine if the reported differential sensitivity to ethanol is reflected in differences in ethanol's effects on cell behavior. Chronic exposure to 200 mM ethanol for 48 hr had a strong inhibitory effect on DNA synthesis in palate cells derived from both the C57BL/6J and ICR strains and a significant effect on protein synthesis in C57BL/6J palate cells. When we attempted to verify strain differences in susceptibility to ethanol teratogenesis, we were not able to observe an increased incidence of birth defects due to ethanol in either strain. High doses of ethanol (5.8 g/kg, administered by intraperitoneal injection on gestational day 8) resulted in death in both C57BL/6J and ICR mice. A lower dose (4.8 g/kg) caused decreased fetal weight and increased resorption in both strains, but did not bring about FAS-like craniofacial dysmorphology in either strain. It appears, therefore, that whereas ethanol can significantly affect the behavior of cells derived from craniofacial tissue, these effects cannot be correlated with sensitivity to ethanol teratogenesis in the mouse system.

Animals↗