Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice.

Endothelial cell progenitors, angioblasts, have been detected in the peripheral blood of adult humans, mice, and rabbits. These cells have been shown to incorporate into the endothelium of newly forming blood vessels in pathological and nonpathological conditions. Here we investigated the possibility that the CD34-expressing leukocytes (CD34(+) cells) that appear to be enriched for angioblasts could be used to accelerate the rate of blood-flow restoration in nondiabetic and diabetic mice undergoing neovascularization due to hindlimb ischemia. CD34(+) cells did not accelerate the restoration of flow in nondiabetic mice, but dramatically increased it in diabetic mice. Furthermore, CD34(+) cells derived from type 1 diabetics produced fewer differentiated endothelial cells in culture than did their type 2 diabetic- or nondiabetic-derived counterparts. In vitro experiments suggest that hyperglycemia per se does not alter the ability of angioblasts to differentiate or of angioblast-derived endothelial cells to proliferate. In contrast, hyperinsulinemia may enhance angioblast differentiation but impair angioblast-derived endothelial cell survival or proliferation. Our findings suggest that CD34(+) cells may be a useful tool for therapeutic angiogenesis in diabetics.

Changes in structure and methylation pattern in a cluster of thymidine kinase genes.

Cell line 101 is a thymidine kinase (TK)-positive derivative of Ltk- which contains ca. 20 copies of the herpes simplex virus TK gene organized in a tandem array. DNA methylation at three sites within the gene and flanking sequences was inversely correlated with expression: the sites were unmethylated in line 101, methylated in each of 4 TK-negative derivatives of 101, and unmethylated in each of 21 TK-positive derivatives derived from them. The same three sites were affected in most of the 20 copies of the TK gene, whereas other sites between them were not affected. Although the entire gene cluster was never lost, indicating that integration into the genome was stable, internal rearrangements occurred at a high frequency. The rearrangements had no obvious correlation with the state of methylation or with the expression of the genes.

In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells.

Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.

Characterization of the mouse JAB1 cDNA and protein.

JAB1 was originally described as a transcriptional coactivator of c-Jun and Jun D. Recent data suggests that JAB1 is a component of a large protein complex, the JAB1 signalosome in mammals and the COP9 complex in plants. The JAB1 signalosome is implicated in the phosphorylation of selected transcription factors, while the COP9 complex is involved in repression of photomorphogenesis in Arabidopsis. In this study, we describe the partial characterization of mouse JAB1 (mJAB1). The murine JAB1 protein is encoded by a gene located on mouse chromosome 1. mJAB1 mRNA is abundantly expressed in a variety of adult tissues as well as in mouse embryos. The JAB1 protein was readily detectable in many cell types and localized to both the nucleus and cytoplasm. Endogenous JAB1 protein is relatively stable and its degradation is not perturbed by blocking 26S proteasome activity, suggesting that this protein is not degraded by the ubiquitin-mediated proteolytic pathway.

Muscle cell differentiation is inhibited by the helix-loop-helix protein Id3.

Id3 (originally named HLH462) belongs to the Id family of the helix-loop-helix transcription factors. Members of the Id family do not contain basic DNA binding regions adjacent to the helix-loop-helix dimerization domain and are, therefore, hypothesized to act as negative regulators of other helix-loop-helix proteins by preventing the formation of functional dimers. We have investigated the potential role of Id3 in the control of muscle cell differentiation. Id3 mRNA is expressed at a high level in proliferating myoblasts and is down-regulated following induction of differentiation. We show that stable overexpression of Id3 mRNA inhibits differentiation of the Sol 8 muscle cell line. Both the HLH and COOH-terminal domains of Id3 are necessary and sufficient for its dominant-negative activity in muscle cells. DNA-binding activity present in nuclear extracts prepared from Id3-overexpressing cells was significantly reduced when compared to the wild-type or vector-transfected cells. Finally, we show by in situ hybridization that the Id3 mRNA is co-expressed with the myogenic regulatory factor myogenin in somites and developing muscle during embryogenesis, although unlike the myogenic regulatory factors, Id3 is also expressed in many other locations in the embryo. These data support a model in which Id3 negatively regulates muscle differentiation by inhibiting the DNA-binding activities of the myogenic regulatory factors.

A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with "zinc finger" sequences.

We have recently identified by cDNA cloning a set of genes that are rapidly activated in mouse 3T3 cells by serum or purified growth factors. Here we report that the cDNA (clone 268) derived from one of these immediate early genes (zif/268) encodes a protein with three tandem "zinc finger" sequences typical of a class of eukaryotic transcription factors. The mRNA of zif/268 is present in many organs and tissues of the mouse and is especially abundant in the brain and thymus tissue. The 5' genomic flanking sequence of zif/268 has sequences related to binding sites for known regulatory proteins, including four sequences that resemble the core of the serum response elements (SREs) upstream of the c-fos and actin genes. The SRE-like sequences could be responsible for the coordinate activation of zif/268 and fos after serum stimulation of 3T3 cells.

Identification of a novel transcriptional activity of mammalian Id proteins.

The Id proteins are a family of related mammalian helix-loop-helix (HLH) proteins which can interact with other HLH proteins but lack a basic region and are thus not thought to bind to DNA. Instead, they are hypothesized to act as dominant negative regulators of DNA-binding basic HLH (bHLH) proteins, by forming inactive heterodimers with these proteins. All four Id family proteins possess related HLH dimerization domains and can interact with similar bHLH proteins, although with differing affinities. The functions of the largely unrelated N- and C-terminal regions of the proteins are unknown. In this study, we have identified a novel transcriptional activity of the mammalian Id proteins. We show that when fused to the heterologous GAL4 DNA-binding domain, all four of the mammalian Id proteins can activate GAL4-dependent transcription. The HLH domain is necessary for the transactivation activity observed, suggesting that interaction with a cellular HLH protein is required. Co-transfection with exogenous Class A bHLH proteins (E-proteins) greatly potentiates the transactivation, which is abolished upon co-transfection with Class B bHLH proteins. These results are consistent with the idea that the Id proteins have a transcriptional activity when present in a DNA-binding complex.

Negative regulation of selected bHLH proteins by eHAND.

The bHLH protein eHAND plays an important role in the development of extraembryonic, mesodermal, and cardiac cell lineages, presumably through heterodimerization with other HLH proteins and DNA binding. In this study, we have identified a novel transcriptional activity of eHAND. In transient transfection assays, eHAND is a potent inhibitor of activation by some but not all bHLH proteins. eHAND can prevent E-box DNA binding by these bHLH proteins. Interestingly, eHAND can also strongly inhibit transactivation activity by a MyoD approximately E47 tethered dimer, which suggests a distinct mechanism of action. eHAND also inhibits MyoD-dependent skeletal muscle cell differentiation and expression of the muscle-specific myosin heavy chain protein. In addition, we show that eHAND can repress activity of the natural p75LNGFR promoter, whose expression overlaps that of eHAND and dHAND. The inhibitory activity of eHAND may be attributed to multiple mechanisms, such as the ability to act as a corepressor, the presence of a repression domain, and its ability to sequester E proteins in an inactive complex. Based upon its inhibitory effect on bHLH proteins and cellular differentiation, we propose that eHAND may function by several mechanisms to promote placental giant cell proliferation by negatively regulating the activities of the bHLH protein MASH-2.

Reciprocal Id expression and myelin gene regulation in Schwann cells.

Id proteins are thought to act as dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors that direct differentiation in various cell types. We found that Schwann cells express all four Id-family genes and that their transcript levels were reciprocally regulated in pairs during nerve maturation in vivo and cAMP-mediated differentiation in vitro. The rapid induction as part of the early response to axonal membranes and cytokines suggested that Id3 is involved in myelin gene repression. An inverse relationship between Id1/3 and myelin P0 expression was consistent with a role for these two Id proteins as inhibitors of differentiation, and Id1/3 proteins strongly repressed myelin gene promoter activity. Nuclear factors isolated from Schwann cells and intact sciatic nerves were found to bind three different HLH recognition sequences (E boxes) in the proximal region of the P0 promoter, and production of these DNA binding complexes was altered during differentiation. These data support the concept that Id proteins regulate myelin gene expression by controlling the formation of specific bHLH DNA binding complexes with different E-box preferences.

Degradation of Id proteins by the ubiquitin-proteasome pathway.

Id proteins act as negative regulators of bHLH transcription factors by forming transcriptionally inactive protein complexes. The proposed function of these proteins includes promotion of cell growth and cell cycle progression, induction of apoptosis, and inhibition of cellular differentiation. We investigated the role of the ubiquitin-mediated proteolytic pathway in the degradation of the Id3 protein. We found Id3 to be a short-lived protein and estimated the half-life to be approximately 20 min in 293 cells. Using specific inhibitors of the 26S proteasome and mutant fibroblast cells with a temperature-sensitive defect in the essential E1 ubiquitin-activating enzyme, we show that Id3 and the related Id1 and Id2 proteins are degraded through the ubiquitin-proteasome pathway. We found the Id4 protein to be much less sensitive to inhibitors of the 26S proteasome, but its degradation was dependent on the E1 enzyme. In addition, we observed that coexpression of the bHLH protein E47 with Id3 significantly reduced the rate of degradation of Id3, suggesting that Id3 is less susceptible to degradation by the 26S proteasome when complexed to a bHLH protein. -Bounpheng, M. A., Dimas, J. J., Dodds, S. G., Christy, B. A. Degradation of Id proteins by the ubiquitin-proteasome pathway.

Differential biological activities of mammalian Id proteins in muscle cells.

Id proteins are helix-loop-helix (HLH) transcription factors that lack DNA-binding domains. These proteins form inactive heterodimers with basic HLH (bHLH) factors, inhibiting their DNA-binding and transcriptional activities. Consistent with a proposed role for Id proteins as inhibitors of terminal differentiation, Id1 and Id3 have been shown to negatively regulate myogenesis in cultured muscle cells. Here we have investigated the possibility that Id2 and/or Id4 can act in a similar manner. Surprisingly, while overexpression of Id2 resulted in inhibition of differentiation of Sol 8 myoblast cells, overexpression of Id4 did not. Sol 8 cells stably transfected with Id4 showed no apparent changes in expression of muscle-specific genes upon differentiation. DNA-binding activities present at the muscle creatine kinase (MCK) enhancer E-box and transcription of the MCK enhancer were not altered in Id4-overexpressing cells, compared with vector-transfected cells. Id2 is also a more potent inhibitor of protein/DNA complex formation at the MCK-R enhancer E-box than Identified in vitro. Therefore, our data support the notion that members of the Id family might be involved in the regulation of distinct developmental pathways.

Convulsant-induced increase in transcription factor messenger RNAs in rat brain.

Administration of the convulsants pentylenetetrazole (Metrazole) or picrotoxin to rats caused a dramatic increase in mRNAs of four putative transcription factor genes, zif/268, c-jun, jun-B, and c-fos, in neurons of the hippocampus and dentate gyrus, as well as other areas of the cerebral cortex, including pyriform cortex and cingulate cortex. The increase in these mRNAs was rapid and transient: amounts peaked within 1 hr and returned to baseline within 2 hr. These results extend the observation made by Morgan et al. [Morgan, J. I., Cohen, D. R., Hempstead, J. L. & Curran, T. (1987) Science 237, 192-197] that c-fos mRNA and protein are induced in rat brain after seizures. We hypothesize that the increase of these putative transcription factor mRNAs in the brain is part of a programmed genomic response of neurons to intense stimulation, which is analogous to the genomic response of nonneuronal cells to growth factors.

Constitutive expression of zif268 in neocortex is regulated by synaptic activity.

Transcription factors are rapidly and transiently induced in brain by excitatory stimuli and may be important in coordinating changes in gene expression underlying neuronal plasticity. In contrast to their transient induction after stimulation, certain transcription factors display stable, relatively high basal levels of expression in brain. Here we demonstrate that this "constitutive" expression of the transcription factor zif268 in cortex is driven by natural synaptic activity. Blockade of afferent visual activity with intraocular injections of tetrodotoxin results in rapid, dramatic reductions of Zif268 mRNA and immunoreactivity in visual cortex. Moreover, dark-adaptation for several days lowers zif268 expression in visual cortex, and expression rapidly returns to control levels upon subsequent light exposure. Several other transcription factors, which are induced in cortical neurons by excitatory stimuli, appear less responsive to changes in natural sensory input. These studies suggest that transcription factors play a role not only in responses to artificial stimuli but also in the normal maintenance of cortical physiology. Anatomic markers for zif268 may be useful in mapping normal cortical activity in brain.

An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene.

An mRNA encoding a helix-loop-helix protein that we have named HLH462 is induced in mouse 3T3 cells as part of the immediate early transcriptional response to growth factors and other signaling agents. The RNA is present in a number of mouse tissues and in the developing mouse fetus. The HLH462 gene has been mapped by interspecific backcross analysis to the distal region of mouse chromosome 4. In its helix-loop-helix region HLH462 is closely related to the Id protein and the Drosophila emc protein. Like Id, HLH462 lacks a basic region required for DNA binding, and it inhibits the DNA-binding activities of other helix-loop-helix proteins. On the basis of its structural and functional similarity to Id, we suggest that HLH462 may inhibit the activities of helix-loop-helix transcription factors during the cellular growth response and during development.

The zinc finger transcription factor Zif268/Egr-1 is essential for Schwann cell expression of the p75 NGF receptor.

Nerve injury alters the function of Schwann cells from quiescent, myelin forming cells to proliferating cells that facilitate nerve repair. The transcription factor, Zif268, may be involved in transmitting injury-related signals since its expression is rapidly induced by nerve transection in vivo and without intervening protein synthesis by injury-related signals in vitro. Expression of the low-affinity p75 nerve growth factor receptor (NGFRp75) by Schwann cells after nerve injury closely correlated with the zif268 expression profile, and Zif268 transactivated the NGFRp75 promoter in transient transfection assays. Conversely, the NGFRp75 gene was not expressed when Zif268 protein was depleted by stable transfection of antisense cDNA. Moreover, nuclear proteins corresponding to Zif268 bound to the NGFRp75 promoter by Southwestern blotting, indicating that a direct interaction of Zif268 with the NGFR gene is required for its expression in Schwann cells.

Expression of the dominant-negative regulator Id4 is induced during adipocyte differentiation.

Expression of the Id4 gene was investigated during differentiation of 3T3-L1 preadipocytes into mature adipocytes. Id4 is a member of a family encoding non-DNA binding helix-loop-helix proteins proposed to inhibit the activity of basic HLH (bHLH) proteins important in many developmental processes. We show here that Id4 expression is low in confluent preadipocytes and rapidly induced by treatment with the combination of hormones which causes differentiation into mature adipocytes. Id4 expression is also induced by treatment with individual hormones, especially dexamathasone. Id4 mRNA can be detected in mouse and human adipose tissue. Genes encoding E-proteins (bHLH proteins known to interact with and be regulated by Id proteins) are expressed and regulated during differentiation in 3T3-L1 cells. These data suggest that the Id4 transcriptional regulator is playing a role in adipose cell differentiation and suggest that DNA-binding HLH proteins may also be important in regulation of differentiation of these cells.

Characterization of natural Epstein-Barr virus infection and replication in smooth muscle cells from a leiomyosarcoma.

Cells from a leiomyosarcoma tumor (LMS-1) from a patient with the acquired immunodeficiency syndrome (AIDS) were explanted, cultured in vitro, and studied by phase-contrast microscopy for morphologic and growth characteristics, immunostaining for cell markers, EBER in situ hybridization and polymerase chain reaction for detection of Epstein-Barr virus (EBV), and immunostaining for expression of EBV antigens. The cells exhibited very slow growth in vitro, with unusual elliptical and spindle-shaped morphology and fragmentation of the cytoplasm into long, tapering, cytoplasmic processes. Greater than 90% of cells expressed diffuse distribution of the smooth muscle isoform of actin by immunoperoxidase staining. Approximately 25% of cells expressed very bright fluorescence by immunostaining of the smooth muscle isoforms of calponin and actin. The majority of cells demonstrated a weak signal for CD21; approximately 5-10% of cells showed a strong signal that was confined to cell surfaces. The cultured cells harbored EBV, and infectious EBV continued to be detected by polymerase chain reaction and virus culture through several passages in vitro. Several EBV antigens were expressed, including latent antigen EBNA-1, immediate-early antigen BZLF1, early antigen EA-D, and late antigens, including viral capsid antigen p160, gp125, and membrane antigen gp350. Human umbilical cord lymphocytes that were transformed with virus isolated from cultured cells yielded immortalized cell lines that expressed EBV antigens similar to other EBV-transformed lymphocyte cell lines. These results confirm that EBV is capable of lytic infection of smooth muscle cells with expression of a repertoire of latent and replicative viral products and production of infectious virus. EBV infection of smooth muscle cells may contribute to the oncogenesis of leiomyosarcomas.

Differential effects on T cell and NK cell development by tissue-specific expression of H-2D(d) transgene.

The effect of tissue-specific expression of the MHC class I molecule H-2D(d) on T cell and NK cell specificity was studied in transgenic mice expressing the H-2D(d) gene under the control of the mouse metallothionein-I promoter. MTD mice expressed high amounts of H-2D(d) in the liver, intestine and testis, but only minute amounts in the thymus, spleen and kidney. Zinc administration resulted in a 1.5- and 8.5-fold increase in H-2D(d) expression in the liver and the intestine, respectively, but did not affect expression in the other organs tested. T cell tolerance developed towards H-2D(d) in MTD mice, even in the absence of zinc. In contrast, NK cell-mediated natural resistance against lymphoma grafts was not seen in MTD mice, despite zinc administration. NK cells in MTD mice also failed to develop self tolerance to H-2D(d). The lack of functional effects did not result from inability of NK cells in MTD mice to interact with H-2D(d), as down-regulation of Ly49A receptor expression was observed on liver NK cells in MTD mice. Our data reveal a difference between T cells and NK cells in their requirements for MHC class I molecules in specificity development.