Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts.

Myogenic cells provide excellent in vitro models for studying the cell growth and differentiation. In this study we report that lysophosphatidic acid (LPA), a bioactive phospholipid contained in serum, stimulates the growth and inhibits the differentiation of mouse C2C12 myoblast cells, in a distinct manner from basic fibroblast growth factor (bFGF) whose mitotic and anti-differentiation actions have been well investigated. These actions of LPA were both blocked by pertussis toxin, suggesting the involvement of Gi class of G proteins, whereas bFGF acts through receptor tyrosine kinases. Detailed analysis revealed that LPA and bFGF act differently in regulating the myogenic basic helix-loop-helix (bHLH) proteins, the key players in myogenic differentiation process. LPA stimulates the proliferation of undifferentiated myoblasts allowing the continued expression of MyoD, but in contrast, bFGF does so with the MyoD expression suppressed at the mRNA level. Both compounds maintain the myf-5 expression, and suppress the myogenin expression. In addition, while LPA did not inhibit cell-cell contact-induced differentiation, bFGF strongly inhibited this process. Furthermore, LPA and bFGF act cooperatively in their mitogenic and anti-differentiation abilities. These findings indicate that LPA and bFGF differently stimulate intracellular signaling pathways, resulting in proliferating myoblasts each bearing a distinct expression pattern of myogenic bHLH proteins and distinct differentiation potentials in response to cell-cell contact, and illustrate the biological significance of Gi-mediated and tyrosine kinase-mediated signals.

Still life, a protein in synaptic terminals of Drosophila homologous to GDP-GTP exchangers.

The morphology of axon terminals changes with differentiation into mature synapses. A molecule that might regulate this process was identified by a screen of Drosophila mutants for abnormal motor activities. The still life (sif) gene encodes a protein homologous to guanine nucleotide exchange factors, which convert Rho-like guanosine triphosphatases (GTPases) from a guanosine diphosphate-bound inactive state to a guanosine triphosphate-bound active state. The SIF proteins are found adjacent to the plasma membrane of synaptic terminals. Expression of a truncated SIF protein resulted in defects in neuronal morphology and induced membrane ruffling with altered actin localization in human KB cells. Thus, SIF proteins may regulate synaptic differentiation through the organization of the actin cytoskeleton by activating Rho-like GTPases.

hikaru genki, a CNS-specific gene identified by abnormal locomotion in Drosophila, encodes a novel type of protein.

We have identified a gene, hikaru genki (hig), whose mutant phenotype includes abnormal locomotor behavior. Mutant first instar larvae have uncoordinated movements, and both larvae and adults have reduced locomotion. Sequence analyses revealed that this gene encodes a novel type of protein with a signal sequence, but without transmembrane regions. One of its domains has similarities with immunoglobulin domains; three or four regions are similar to a complement-binding domain found in complement-related proteins and selectins. In situ hybridization to embryos revealed that accumulation of the hig transcripts is restricted to subsets of cells in the CNS. Our data suggest that hig has a role in the development of CNS functions involved in locomotor activity.

Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons.

Distinct classes of neurons are generated at defined times and positions during development of the nervous system. It remains elusive how specification of neuronal identity coordinates with acquisition of pan-neuronal properties. Here we show that basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Olig2 and Ngn2 are specifically coexpressed in motoneuron progenitors. Misexpression studies in chick demonstrate the specific, combinatorial actions of Olig2 and Ngn2 in motoneuron generation. Our results further revealed crossregulatory interactions between bHLH and homeodomain transcription factors in the specification of motoneurons. We suggest that distinct classes of transcription factors collaborate to generate motoneurons in the ventral neural tube.

Regulation of the chicken embryonic myosin light-chain (L23) gene: existence of a common regulatory element shared by myosin alkali light-chain genes.

The transcriptional regulation of the chicken myosin alkali light-chain (MLC) L23 gene was analyzed. Two different types of cis-regulatory regions were identified: one was a silencerlike region located between 3.7 and 2.7 kilobases upstream of the mRNA initiation site, and the other was essential for the expression of L23 in skeletal muscle cells and was located between 106 and 91 base pairs upstream of the cap site. This 16-base-pair cis-acting element was designated as the MLC box since it is well conserved in various muscle-specific MLC promoter regions. The activity of the MLC box showed tissue specificity. To analyze the relationship between the nucleotide sequence and the activity of the MLC box precisely, mutation analysis was performed. The 16-base-pair sequence was indispensable for the active transcription of L23 gene, and the MLC box could function in either orientation. The inverted sequence of the MLC box was similar to the sequence of the alpha-actin CArG box. By using a gel mobility retardation assay, the nuclear protein(s) that binds to both MLC box and CArG box was detected with nuclear extract prepared from chicken embryonic breast muscle. These observations imply that a common factor regulates the coordinate expression of these contractile proteins in muscle differentiation.

MyoD and myogenin act on the chicken myosin light-chain 1 gene as distinct transcriptional factors.

Expression of MyoD, myogenin, MRF4, and Myf-5 converts nonmuscle cells to muscle cells. In an attempt to analyze the roles of these factors, we have investigated their effects on transcription driven by the promoter of the chicken myosin alkaline light-chain (MLC1) gene. The activation by CMD1 or c-myogenin (chicken MyoD or myogenin, respectively) was dependent on the existence of a muscle-specific regulatory region located from positions -2096 to -1743. Its distal half, containing a pair of E boxes (CANNTG), had been previously characterized as an enhancer responsive to CMD1 but not to c-myogenin. In this study, we report the identification of another enhancer in the muscle-specific regulatory region which is preferentially responsive to c-myogenin. Deletion and mutation analyses indicated that this enhancer requires a single E box and its flanking sequences. Furthermore, analysis of chimeric proteins of CMD1 and c-myogenin indicated that regions outside the basic helix-loop-helix domain of c-myogenin are involved in the specificity of the enhancer. These results show that CMD1 and c-myogenin act on the MLC1 gene by recognizing different upstream DNA sequences and that direct or indirect interactions between the regions outside the basic helix-loop-helix domain and flanking sequences of E boxes are involved in the target sequence specificity.

Upstream regulatory region for inducible expression of the chicken skeletal myosin alkali light-chain gene.

The expression of the fast type of myosin alkali light chain 1 is induced during the differentiation of muscle cells. To study the mechanism of its gene regulation, we joined the sequence of the 5'-flanking and upstream region of the chicken myosin alkali light-chain gene to the structural gene for chloramphenicol acetyltransferase (CAT). The fusion gene was introduced either into quail myoblasts transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsNY68) or into chicken myoblasts, and the transiently expressed CAT activity was assayed after the differentiation of the myoblasts. From the experiments with the external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in response to the cell differentiation was found to be localized at 2 kilobases upstream of the transcription initiation site. This region of 160 nucleotides contained two pairs of short sequences worthy of note, a direct repeat of 12 nucleotides, and an inverted repeat of 8 nucleotides. The nucleotide sequences of the 5'-flanking sequence up to nucleotide -3381 were determined and compared with those of the upstream activating elements of actin genes.

The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation.

The repressor delta EF1 was discovered by its action on the DC5 fragment of the lens-specific delta 1-crystallin enhancer. C-proximal zinc fingers of delta EF1 were found responsible for binding to the DC5 fragment and had specificity to CACCT as revealed by selection of high-affinity binding sequences from a random oligonucleotide pool. CACCT is present not only in DC5 but also in the E2 box (CACCTG) elements which are the binding sites of various basic helix-loop-helix activators and also the target of an unidentified repressor, raising the possibility that delta EF1 accounts for the E2 box repressor activity. delta EF1 competed with E47 for binding to an E2 box sequence in vitro. In lymphoid cells, endogenous delta EF1 activity as a repressor was detectable, and exogenous delta EF1 repressed immunoglobulin kappa enhancer by binding to the kappa E2 site. Moreover, delta EF1 repressed MyoD-dependent activation of the muscle creatine kinase enhancer and MyoD-induced myogenesis of 10T1/2 cells. Thus, delta EF1 counteracts basic helix-loop-helix activators through binding site competition and fulfills the conditions of the E2 box repressor. In embryonic tissues, the most prominent site of delta EF1 expression is the myotome. Myotomal expression as well as the above results argues for a significant contribution of delta EF1 in regulation of embryonic myogenesis through the modulation of the actions of MyoD family proteins.

Lack of effective messenger RNA for beta 2-microglobulin in a gestational human choriocarcinoma cell line (GCH-1).

When two lines of gestational human choriocarcinoma cells (GCH-1 and GCH-2) were incubated with [35S]methionine, the labeled beta 2-microglobulin was not detected on sodium dodecyl sulfate:polyacrylamide gel electrophoresis after purification of the labeled protein with affinity chromatography, using rabbit antibody against beta 2-microglobulin as the ligand. In contrast, in the case of nongestational choriocarcinoma cells originating from male stomach cells, the incorporation of [35S]methionine into beta 2-microglobulin was observed. When polyadenylate-containing messenger RNA prepared from GCH-1 cells was incubated with [35S]methionine in a rabbit reticulocyte lysate system, the labeling of beta 2-microglobulin was not shown. On the other hand, when polyadenylate-containing messenger RNA from nongestational choriocarcinoma cells was incubated in the same cell-free system, labeled beta 2-microglobulin was detected. These results indicate that beta 2-microglobulin is not synthesized in gestational human choriocarcinoma cells and at least in the case of GCH-1, beta 2-microglobulin is not synthesized owing to the lack of effective messenger RNA for beta 2-microglobulin.

Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord.

Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cell-surface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.

Biosynthesis of ribosomal proteins by poly(A)-containing mRNAs from rat liver in a wheat germ cell-free system and sizes of mRNAs coding ribosomal proteins.

(1) Poly(A)-containing mRNAs from total polysomal RNA of regenerating rat liver were incubated with [3H]leucine in a wheat germ cell-free system. Ribosomal proteins were purified as described previously [1], and with two-dimensional gel electrophoresis. The proteins on the gel except for less basic protein had appreciable radioactivity, whereas the surrounding areas had very low radioactivity. Acetic acid-soluble proteins labeled in this system were subjected to three-dimensional gel electrophoresis [2]. Except for L1 and L2 proteins, each of the ribosomal proteins, including less basic ones, showed a major radioactive peak coinciding with the protein band on SDS gel. Thus, the wheat germ cell-free system completely translates almost all mRNAs for individual ribosomal proteins. Equimolar amounts of almost all ribosomal proteins were synthesized in the presence of the saturating concentration of mRNAs. (2) Free polysomes from regenerating rat liver were fractionated into three sizes. Each class of polysomes was incubated with [3H]leucine. Ribosomal proteins with molecular weights of 40 000 to 21 000 were mainly synthesized by Fraction B (5-14 monomeric ribosomes), L1 and L2 [2] with 60 000 and 54 000, by Fraction C (greater than 15 monomeric ribosomes) and B, and ribosomal proteins smaller than 20 000 by Fractions A (less than pentamer) and B. (3) mRNAs from rat liver total polysomes were fractionated into seven classes by size and each was translated in the wheat germ extract. Ribosomal proteins with molecular weights of 54 000 to 30 000 were mainly synthesized by mRNAs of 12 to 14.5 S, ribosomal proteins of 35 000 to 22 000 by those of 9.5 to 12 S, ribosomal proteins of 22 000 to 13 000 by those of 7 to 9.5 S, and smaller ribosomal proteins by those smaller than 7 S. These results indicate that individual ribosomal proteins are synthesized by monocistronic mRNAs, the lengths of which are proportional to the molecular weights of the corresponding ribosomal proteins.

Preferential biosynthesis of ribosomal structural proteins by free and loosely bound polysomes from regenerating rat liver.

Homologous cell-free systems were prepared using free, total bound, tightly bound or KCl-sensitive loosely bound (KCl-sensitive) polysomes from regenerating rat liver. [14C]Leucine was incubated with one kind of polysomes and [3H]leucine with another kind. The reaction mixtures were then combined, and ribosomal structural proteins were purified as described previously [4], using two-dimensional polyacrylamide gel electrophoresis as the final step [5]. The 3H to 14C ratios of the purified fractions were estimated to compare the activities of the two kinds of polysomes for biosynthesis of ribosomal structural proteins. The following results were obtained: (1) The activity of free polysomes for biosynthesis of ribosomal structural proteins was about 3.6 or 2.4 times higher than that of total bound polysomes in two experiments in which 14C and 3H labeling was reversed. The radioactivities incorporated by free polysomes into most of the proteins separated on two-dimensional gel were found to be definitely higher than those in the surrounding areas, suggesting that most of the ribosomal structural proteins were synthesized by free polysomes. The activity of free polysomes for biosynthesis of ribosomal structural proteins was about 7 times higher than that of tightly bound polysomes, which were prepared by washing the microsomal membrane fraction with 0.5 M KCl. The radioactivities incorporated by tightly bound polysomes into the proteins separated on two-dimensional gel were only slightly higher than those in the surrounding areas, indicating that these polysomes had very low synthetic activity. (2) Preferential synthesis of histones by free polysomes was also shown using the same procedures. (3) KCl-sensitive polysomes which were released by washing the microsomal membrane fraction with 0.5 M KCl, were shown to have definitely higher activity than tightly bound polysomes for biosynthesis of ribosomal structural proteins. (4) From these results, it is concluded that most of the ribosomal structural proteins are preferentially synthesized by free and KCl-sensitive polysomes in regenerating rat liver.

Single chicken cardiac myosin alkali light-chain gene generates two different mRNAs by alternative splicing of a complex exon.

We have isolated and characterized two kinds of cDNA for the chicken cardiac myosin alkali light chain. The sequences of the two cDNAs are identical, except for a notable divergence in part of the 3' untranslated sequence. By analysis of isolated genomic clones, it was shown that the genomic sequences corresponding to the different sequences in the 3' untranslated regions of the two mRNAs were arranged within a limited part of a single stretch of DNA; also the two distinct 3' untranslated regions of the two mRNAs shared part of the last exon, which was 0.6 x 10(3) base-pairs long. There are two canonical acceptor sites available for RNA splicing in the last exon, the first being located at the 5' end of the exon, and the second at 370 base-pairs downstream from this end. Together with analysis by S1 nuclease mapping, the foregoing results lead us to conclude that, by the differential use of these two acceptor sites, a single gene generates two distinct mRNAs of 1.45 x 10(3) base-pairs and 1.1 x 10(3) base-pairs with or without the 5' half of the last exon. The two mRNAs appear to utilize the same modified poly(A) signal, AGTAAA, rather than the authentic AATAAA sequence present about 30 base-pairs downstream from the poly(A) attachment sites. This is probably because another consensus G + T-rich sequence is present at an appropriate distance from the AGTAAA sequence, but not from the AATAAA sequence. The gene for the cardiac myosin alkali light chain has proved to be expressed in ventricular muscle and in atrial and anterior latissimus dorsi muscles, the last of these being characteristic of slow skeletal muscle. In these muscles, two kinds of mRNA for the cardiac myosin alkali light chain, identical with those in ventricular muscle, were expressed and their relative amount in each tissue was almost the same as that in ventricular muscle.

Isolation of the chick myosin alkali light chain gene expressed in embryonic gizzard muscle and transitional expression of the light chain gene family in vivo.

A chick embryonic myosin alkali light chain L23 gene that is expressed transiently at embryonic stages in chick skeletal, cardiac and smooth muscles and in brain continuously from embryo to adult stages, was isolated and characterized. Sequence analysis showed that the exonic sequence of this gene was identical with that of embryonic myosin light chain mRNA except for one base replacement. This gene is a single gene of 5200 bases, which is divided into seven exons by six introns, and the positions of inserts of all the introns are well-conserved as in the skeletal and cardiac muscle myosin alkali light chain genes. Therefore, this embryonic myosin light chain gene can be classified as a member of the myosin alkali light chain gene family, and these three genes may have originated from a common ancestral gene. Transcription of the embryonic light chain gene starts from the same initiation site 33 bases upstream from ATG in embryonic muscle tissues and brain. Comparison of the nucleotide sequence around the promotor region of the embryonic myosin light chain gene with the corresponding regions of the skeletal and cardiac myosin light chain genes showed that the 11-base consensus sequence (TCCTATTTATAG) is present about 100 bases upstream from the transcription initiation site in each gene.

Spatially- and temporally-restricted expression of meltrin alpha (ADAM12) and beta (ADAM19) in mouse embryo.

The cloning of the full-length cDNA encoding meltrin beta (ADAM19), one of the metalloprotease-disintegrins expressed in mouse myogenic cells, revealed that the meltrin beta gene encodes a membrane protein closely related to meltrin alpha (ADAM12) which participates in myotube formation in vitro. To delineate the functions of meltrin alpha and beta, we examined the expression patterns of their transcripts during embryogenesis. The meltrin alpha gene is activated in condensed mesenchymal cells that give rise to skeletal muscle, bones and visceral organs. Meltrin beta mRNA, in contrast, is markedly expressed in craniofacial and dorsal root ganglia and ventral horns of the spinal cord, where peripheral neuronal cell lineages differentiate. Heart, skeletal muscle, intestine and lung also express meltrin beta mRNA transiently. Although the meltrin alpha and beta transcripts exhibit distinct expression patterns during embryogenesis, both genes are mainly activated in mesenchymal cells that are derived from both mesoderm and ectoderm.

Ets family transcription factor ESE-1 is expressed in corneal epithelial cells and is involved in their differentiation.

Involvement of an epithelium-specific transcription factor ESE-1/ESX/ELF3/jen (ESE-1) in corneal epithelial cell differentiation was investigated. ESE-1 was reported to be induced during terminal differentiation of the epidermis and primary keratinocytes and to transactivate target genes through ets binding sites. However, its expression and function in corneal epithelium have not been examined. We report here that ESE-1 is upregulated upon differentiation in mouse corneal epithelium and in immortalized human corneal epithelial cells (HCE). ESE-1 transactivates through the regulatory element of cornea-specific K12 keratin. Moreover, introduction of ESE-1 antisense RNA in HCE cells affect their differentiation. These data suggest the involvement of ESE-1 in differentiation of corneal epithelial cells.

Molecular cloning and expression analyses of mouse betaklotho, which encodes a novel Klotho family protein.

We report here the identification of mouse betaklotho (betakl), which encodes a type I membrane protein with high resemblance to Klotho (KL). Both betaKL and KL consist of two internal repeats with homology to family 1 glycosidases, while these essential glutamates for the enzymatic activities were not conserved. The identical pattern of substitution and variation in the substituted amino acids between these two proteins indicate that they likely to form a unique family within the glycosidase family 1 superfamily. During mouse embryonic development, strong betakl expression was detected in the yolk sac, gut, brown and white adipose tissues, liver and pancreas, and in the adult, predominantly in the liver and pancreas. Despite the high structural similarity between betaKL and KL, their expression profiles were considerably different and betakl expression was not induced in kl-deficient mouse mutants.

Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3.

Basic helix-loop-helix (bHLH) transcription factors have been shown to be essential for specification of various cell types. Here, we describe a novel bHLH family consisting of three members, two of which (Olig1, Olig2) are expressed in a nervous tissue-specific manner, whereas the third, Olig3 is found mainly in non-neural tissues. Olig1 and Olig2, which recently have been implicated in oligodendrogenesis, are expressed in the region of the ventral ventricular zone of late embryonic spinal cord where oligodendrocyte progenitors appear. In the embryonic brain, the Olig2 expression domain is broader than that of Olig1 and does not overlap with an oligodendrocyte progenitor marker, CNP. Furthermore, Olig2 is expressed in most cells in the ventral half of the early embryonic spinal cord, which do not yet express an early neuronal marker TuJ1. These results indicate that Olig2 expression is not limited to the oligodendrocyte lineage but includes immature neuronal progenitors and multipotential neuron/glia progenitors as well as embryonic olfactory neurons.

Molecular cloning and expression of a novel klotho-related protein.

Klotho protein is a novel beta-glucosidase-like protein produced predominantly in the kidney. The klotho mouse, which genetically lacks klotho gene expression, manifests various systemic phenotypes resembling aging. In the present study we succeeded in isolating a novel human protein structurally related to klotho protein. The protein possesses one beta-glucosidase-like domain and is 42% identical with klotho protein at the amino acid level. Unlike klotho protein, it possesses neither a signal sequence nor a transmembrane domain, suggesting that it is a cytosolic protein, and thus was termed cytosolic beta-glucosidase-like protein-1 (cBGL1). By Northern blot analysis cBGL1 mRNA was expressed most abundantly in the liver, followed by the small intestine, colon, spleen, and kidney. When klotho and cBGL1 gene expression was examined in renal cell carcinoma tissues, both klotho and cBGL1 mRNA levels in tumors were lower than those in nontumor regions, suggesting that renal epithelial cells may lose klotho and cBGL1 gene expression during the course of malignant transformation. In conclusion, we describe the primary structure and gene expression of a novel protein related to klotho protein.

Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice.

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.