Involvement of Gas7 along the ERK1/2 MAP kinase and SOX9 pathway in chondrogenesis of human marrow-derived mesenchymal stem cells.

OBJECTIVE: The growth-arrest-specific protein, Gas7, has been shown to be involved in reorganization of the cytoskeleton and for inducing changes in cell shape during cell differentiation. The goals of this study were to investigate the novel role of human Gas7 (hGas7) in chondrogenic differentiation of human mesenchymal stem cells (hMSCs) and to identify the relationship between hGas7, extracellular signal-regulated kinase (ERK1/2) and SOX9 in the chondrogenic pathway. METHODS: Bone marrow-derived hMSCs were induced to undergo chondrogenic differentiation with transforming growth factor-beta1 (TGF-beta1) in an aggregate culture system. The expression of hGas7 and SOX9 and phosphorylation of ERK1/2 at multiple time points were investigated. Chondrogenic capacity was evaluated by the size of aggregates, by glycosaminoglycan content, and by type II collagen and proteoglycan deposition after interfering with expression of hGas7, ERK1/2 or SOX9. To delineate the functional role of these genes in chondrogenesis, inhibition of individual gene's expression in hMSCs, by antisense oligonucleotides or interference RNA (siRNA), and the effect on chondrogenic differentiation were also investigated. RESULTS: Treatment of hMSCs with TGF-beta1 resulted in a transient up-regulation of hGas7b, one of the hGas7 isoforms (day 3-day 5), a transient phosphorylation of ERK1/2 (0.5-4 h) and an up-regulation of SOX9 (2 h to day 14). Transient expression of hGas7b was also detected in hMSCs by reverse transcription-polymerase chain reaction at day 2 and day 3 following TGF-beta1 treatment. Interference with hGas7b production by hGas7b-specific antisense oligonucleotide or inhibition of p-ERK with PD98059, a specific inhibitor of ERK signaling pathway, or interference with SOX9 production by SOX9 siRNA all caused adverse effects of chondrogenic differentiation of hMSCs. Meanwhile, inhibition of p-ERK or SOX9 both blocked the expression of hGas7b. However, the p-ERK and SOX9 pathway was not affected by inhibition of hGas7b. CONCLUSION: These results provide evidence that the transient expression of hGas7b, regulated by activation of ERK1/2 and SOX9 pathway, is essential for chondrogenic differentiation of hMSCs.

Humic acid induced growth retardation in a sertoli cell line, TM4.

Humic acid (HA) is a fluorescent deep brown organic, polymeric compound composed of phenolic acid. Intraperitoneal injection of HA in rats induced testicular morphological changes including degeneration of the seminiferous tubule, reduction in the number of Sertoli cells and spermatogonia, and a loss of spermatids. It was suggested that Sertoli cells may be involved in the progression of testicular atrophy. In this study, we used a mouse Sertoli cell Line, TM4, to investigate the effect of HA on Sertoli cells and the mechanism of the testicular atrophy induced by HA. We found that the cell growth of TM4 cells were reduced in 1 to 4 days of HA exposure. FACScan analysis of the DNA content of HA-treated TM4 cells revealed that there was no sub-G1 peak, indicating that the TM4 cells did not commit to the programmed cell death. However, a large proportion of TM4 cells were arrested at the G1 phase. The percentage of TM4 cells at the G1 phase increased from 36% to 84% after HA treatment for 4 days. Western blot assay of HA-treated TM4 cells showed that the expression of cyclin D1 protein decreased while the expression of p27kiP1 protein increased. These results suggest that HA-induced testicular atrophy is linked in part to an inhibitory effect on the growth of Sertoli cells. This model may be useful in investigation of environmental agents inducing testicular atrophy.

RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E.

RNase E isolated from Escherichia coli is contained in a multicomponent "degradosome" complex with other proteins implicated in RNA decay. Earlier work has shown that the C-terminal region of RNase E is a scaffold for the binding of degradosome components and has identified specific RNase E segments necessary for its interaction with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase. Here, we report electron microscopy studies that use immunogold labeling and freeze-fracture methods to show that degradosomes exist in vivo in E. coli as multicomponent structures that associate with the cytoplasmic membrane via the N-terminal region of RNase E. Whereas PNPase and enolase are present in E. coli in large excess relative to RNase E and therefore are detected in cells largely as molecules unlinked to the RNase E scaffold, immunogold labeling and biochemical analyses show that helicase is present in approximately equimolar amounts to RNase E at all cell growth stages. Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E. coli, also suggest that RNA processing and decay may occur at specific sites within cells.

The Gas7 gene encodes two protein isoforms differentially expressed within the brain.

Gas7, a growth arrest-specific gene first isolated from serum-starved NIH3T3 cells, is expressed abundantly in the brain and is essential for the outgrowth of neurites from cultured cerebellar neurons. Here, we report the existence of a Gas7-related cDNA, designated Gas7-cb, isolated from the mouse cerebellum, and we report the finding that Gas7-cb transcripts and protein are expressed at different locations than those of Gas7. Gas7-cb cDNA differs from the Gas7 cDNA only in the 5' region. Its encoded protein shares the same 320 amino acids in its C-terminus with those of Gas7. Analyses of the RNA and protein expression of Gas7-cb and Gas7 by RNase protection assay and Western blot indicated that while Gas7 expression is predominant in the cerebrum and in growth-arrested NIH3T3 fibroblasts, Gas7-cb expression is predominant in the cerebellum. Characterization of Gas7 and Gas7-cb RNAs and of the genomic structure of murine Gas7 cloned in a bacterial artificial chromosome indicated that the Gas7 gene spans more than 60 kb and consists of at least 15 exons. The 5'-terminus of Gas7-cb is located at exon 6a, which is absent in Gas7 transcripts but is retained in its entirety in Gas7-cb transcripts, resulting in the presence of a unique 20-amino-acid sequence at the N-terminus of the Gas7-cb protein. Our results show that the Gas7 gene encodes two Gas7 isoforms, Gas7 and Gas7-cb, whose expression is differentially regulated within mouse brain.

RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity.

During recent studies of ribonucleolytic "degradosome" complexes of Escherichia coli, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is ssrA (for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt ssrA RNA is generated by RNase E cleavage at the CCA-3' terminus of a 457-nt ssrA RNA precursor and that interference with this cleavage in vivo leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature ssrA RNA and for normal ssrA RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in E. coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3' end of ssrA RNA.

The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly.

Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components.

gas7: A gene expressed preferentially in growth-arrested fibroblasts and terminally differentiated Purkinje neurons affects neurite formation.

Growth arrest-specific (gas) genes are expressed preferentially in cells that enter a quiescent state. gas7, which we identified in serum-starved murine fibroblasts, is reported here to be expressed in vivo selectively in neuronal cells of the mature cerebral cortex, hippocampus, and cerebellum. gas7 transcripts encode a 48-kDa protein containing a structural domain that resembles sequences of OCT2, a POU transcription factor implicated in neuronal development, and synapsins, which have a role in modulating neurotransmitter release. Using in situ hybridization and immunocytochemical analysis, we show that GAS7 expression occurs prominently in cerebellar Purkinje cells and that inhibition of production in terminally differentiating cultures of embryonic murine cerebellum impedes neurite outgrowth from maturing Purkinje cells. Conversely, GAS7 overexpression in undifferentiated neuroblastoma cell cultures dramatically promotes neurite-like outgrowth. Collectively, our results provide evidence for an association between expression of this gas gene and neuronal development.

RNA components of Escherichia coli degradosome: evidence for rRNA decay.

Recently, we found that a multicomponent ribonucleolytic degradosome complex formed around RNase E, a key mRNA-degrading and 9S RNA-processing enzyme, contains RNA in addition to its protein components. Herein we show that the RNA found in the degradosome consists primarily of rRNA fragments that have a range of distinctive sizes. We further show that rRNA degradation is carried out in the degradosome by RNase E cleavage of A+U-rich single-stranded regions of mature 16S and 23S rRNAs. The 5S rRNA, which is known to be generated by RNase E processing of the 9S precursor, was also identified in the degradosome, but tRNAs, which are not cleaved by RNase E in vitro, were absent. Our results, which provide evidence that decay of mature rRNAs occurs in growing Escherichia coli cells in the RNA degradosome, implicate RNase E in degradosome-mediated decay.

Processing of the rne transcript by an RNase E-independent amino acid-dependent mechanism.

RNase E is encoded by the rne (also known as ams or hmp) gene and is the principal enzyme that controls the chemical decay of bulk mRNA in Escherichia coli. Earlier work has shown that RNase E degrades its own mRNA, autoregulating production of the RNase E protein. Here we show that in cells lacking RNase E activity, the 3.6-kilobase rne gene transcript is cleaved site specifically at two locations near its center by a novel endonuclease whose activity is modulated by the presence or absence of amino acids in the culture medium. These cleavages produce a 2-kilobase RNase E-sensitive RNA fragment corresponding to the 3' half of the transcript. Using primer extension and RNase protection analysis, we mapped RNase E-independent cleavages to sites 1558 and 1576 nucleotides from the 5' end of the rne transcript (coordinates 1738 and 1747 of the rne gene). Our results indicate the existence of a previously unknown RNase E-independent mechanism for degradation of rne transcripts and further demonstrate that this mechanism responds to changes in cell growth conditions.

Population cell differentiation of Serratia marcescens on agar surface and in broth culture.

The bacterium Serratia marcescens shows population surface migration (swarming) phenomenum on an LB swarming plate, and differentiated cells can be observed at the swarming front. How the cell population differentiates during swarming on the agar surface is not known, neither is it clear whether cells with differentiated characteristics can be observed in broth culture. To monitor the population cell differentiation in a highly sensitive way without cell destruction, experiments were designed using bacterial luciferase genes luxAB as the reporter genes to allow direct monitoring of the differentiating cells through bioluminescence. An isogenic S. marcescens strain was constructed with luxAB under the control of the promoter of flagellin gene hag (phag::luxAB). Patterns of cell differentiation were monitored either by direct X-ray film exposure and/or by Autolumat luminometer detection. Results show that population cell differentiation on the agar surface occurs first in a temporal and then spatial way during colonial growth. It was also found that cells harvested from both the spreading agar plate and broth culture showed differentiation patterns similar to those from swarming cells, suggesting that the agar surface culture may not be essential for the formation of differentiated cells.

The platelet-derived growth factor alpha-receptor is encoded by a growth-arrest-specific (gas) gene.

Using the Escherichia coli lacZ gene to identify chromosomal loci that are transcriptionally active during growth arrest of NIH 3T3 fibroblasts, we found that an mRNA expressed preferentially in serum-deprived cells specifies the previously characterized alpha-receptor (alphaR) for platelet-derived growth factor (PDGF), which mediates mitogenic responsiveness to all PDGF isoforms. Both PDGFalphaR mRNA, which was shown to include a 111-nt segment encoded by a DNA region thought to contain only intron sequences, and PDGFalphaR protein accumulated in serum-starved cells and decreased as cells resumed cycling. Elevated PDGFalphaR gene expression during serum starvation was not observed in cells that had been transformed with oncogenes erbB2, src, or raf, which prevent starvation-induced growth arrest. Our results support the view that products of certain genes expressed during growth arrest function to promote, rather than restrict, cell cycling. We suggest that accumulation of the PDGFalphaR gene product may facilitate the exiting of cells from growth arrest upon mitogenic stimulation by PDGF, leading to the state of "competence" required for cell cycling.

RNase E cleaves at multiple sites in bubble regions of RNA I stem loops yielding products that dissociate differentially from the enzyme.

Earlier work has shown that RNase E cleaves RNAI, the antisense repressor of replication of ColE1-type plasmids, producing pRNAI-5, whose further decay is mediated by the poly(A)-dependent activity of polynucleotide phosphorylase and other 3' to 5' exonucleases. Using a poly(A) polymerase-deficient strain to impede exonucleolytic decay, we show that RNAI is additionally cleaved by RNase E at multiple sites, generating a series of decay intermediates that are differentially retained by the RNA binding domain (RBD) of RNase E. Primer extension analysis of RNAI decay intermediates and RNase T1 mapping of the cleavage products of RNAI generated in vitro by affinity-purified RNase E showed that RNase E can cleave internucleotide bonds in the bubble regions of duplex RNA segments and in single-stranded regions. Chemical in situ probing of a complex formed between RNAI and the RBD indicates that binding to the RBD destabilizes RNAI secondary structure. Our results suggest a model in which a series of sequential RNase E-mediated cleavages occurring at multiple sites of RNAI, some of which may be made more accessible to RNase E by the destabilizing effects of its RBD, generate RNA fragments that are further degraded by poly(A)-dependent 3' to 5' exonucleases.

Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.

Characterization of the structure and melting behavior of the loop I fragment of ColE1 RNA I.

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5'-end, and a 31-mer which has 11 5'-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5' end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32 degrees C and 71 degrees C clearly discernible. In conjunction with NMR melting study the major transition at 71 degrees C is assigned to the overall melting of the stem region and the 32 degrees C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.

Induction, not associated with host-cell re-activation of damaged plasmid DNA, of damaged-DNA-recognition proteins by retinoic acid and dibutyryl cyclic AMP in mammalian cells.

Our previous studies [Chao (1992) Biochem. J. 282, 203-207; C.C.-K. Chao, unpublished work] has suggested a correlation between the levels of constitutive UV-damaged-DNA-recognitionproteins (UVDRP) and cellular DNA repair in different cell types. In the present study, UVDRP were induced in F9 and NIH3T3 cells by 0.1 microM retinoic acid (RA) and 1 mM dibutyryl cyclic AMP (dbcAMP), which is sufficient to induce differentiation in murine F9 stem cells. The induction of UVDRP in F9 and NIH3T3 cells was optimized after 6 and 2 days incubation with RA/dbcAMP respectively. Since NIH3T3 cells were not induced to differentiate by RA/dbcAMP, the upregulation of the UVDRP in mammalian cells would thus seem not to be mediated directly by differentiation. Using a plasmid re-activation assay to estimate DNA repair, we did not find a correlation between DNA repair and UVDRP in RA/dbcAMP-treated cells. The results suggest that UVDRP may have a function other than, or in addition to, its role in DNA repair.

Rapid identification and isolation of transcriptionally active regions from mouse genomes.

We report here the design, construction and testing of a self-inactivating (Sin) retrovirus promoter-trap vector suitable for identifying and isolating transcriptionally active regions from the mouse genome. When this vector, which contains the bacterial aph gene as its reporter, is integrated into a site downstream from an active host cell promoter, it expresses aph, whose product, aminoglycoside phosphotransferase, produces resistance to the antibiotic G418 in mammalian cells. The construct also contains a native aph promoter which functions in bacteria, but not in mouse cells, to express kanamycin (Km) resistance, plus an adjacent pBR322-derived replication origin. Thus, mammalian DNA segments containing actively transcribed regions flanking aph can be quickly isolated by restriction endonuclease treatment of total DNA from provirus-containing mouse cells, followed by self-ligation, transformation and Km selection of plasmids carried by bacteria transformed with this DNA. We tested this Sin retrovirus promoter-trap system by isolating eight DNA segments upstream to the provirus integration sites in the genome of virus-infected mouse F9 cells. We found that the Sin retrovirus vector produces a high yield of infectious virus particles carrying aph, and that the isolated genomic DNA fragments of F9 cells are transcriptionally active.

RNAII transcribed by IPTG-induced T7 RNA polymerase is non-functional as a replication primer for ColE1-type plasmids in Escherichia coli.

RNAII, an RNA species encoded by ColE1-type plasmids, serves as a primer for plasmid DNA replication. Previous work has shown that overproduction of RNAII transcribed by Escherichia coli RNA polymerase results in elevated plasmid copy number. To produce a plasmid in which the elevation of its copy number is inducible, we placed transcription of RNAII under the control of a bacteriophage T7 late promoter regulated by IPTG-inducible T7 RNA polymerase. During induction of T7 RNA polymerase by IPTG, we found that RNAII was overexpressed, but that, surprisingly, this increase in RNAII did not result in elevation of plasmid copy number. These results suggest that RNAII transcribed by T7 RNA polymerase does not function as a primer for plasmid DNA replication. Since RNAII function requires folding of its multiple stem-loop structures in a precise conformation and folding of RNAII can be influenced by its rate of transcription, the extremely rapid rate of travel of the T7 RNA polymerase may preclude proper folding of RNAII during its elongation.

Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops.

The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.

A+U content rather than a particular nucleotide order determines the specificity of RNase E cleavage.

Ribonuclease E has a central role in Escherichia coli mRNA decay and is dependent on a functional product of the rne (also called ams or hmp1) gene. We investigated the requirements for RNase E cleavage by introducing random mutations into the decanucleotide region at the 5' end of pACYC184 RNA I and studying the effects of these mutations on the position of rne-dependent cleavage in vivo and RNase E-mediated cutting in vitro. We find that the precise point of RNase E cleavage can be altered specifically and reproducibly by sequence changes in the region cleaved and, therefore, is not determined by a distance measured in nucleotides from any other sequence or region of secondary structure in RNA I. Although cleavage by RNase E occurs within sequences rich in A and/or U nucleotides and is affected by the extent of continuity of A and U nucleotides in the regions cleaved, there is no simple relationship between the order of nucleotides and the phosphodiester bond cleaved. Thus, our results are not consistent with either the notion that RNase E cleavages are determined by a simple consensus sequence or the contrary view that RNase E has few primary structural constraints other than a preference for cleaving 5' to an AU dinucleotide.

Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid.

RNase E, an endoribonuclease encoded by the Escherichia coli ams/rne/hmp1 locus, cleaves RNA I, an antisense regulator of the replication of ColE1 type plasmids, in a single-stranded region near its 5' end. The rne-3071 mutation prolongs the RNA 1 half-life in cells cultured at an elevated temperature and imparts temperature sensitivity on RNase E isolated from the mutant strain. Here we report the effects of specific sequence changes introduced by site-directed mutagenesis on the location of ribonucleolytic cleavage near the 5' end of pBR322 RNA I in rne-3071 and congenic rne+ E. coli and on cleavage of RNA I by RNase E in vitro. Primer extension analyses showed that the occurrence and position of cleavages in vivo and in vitro are altered highly specifically by sequence changes but that the site of cleavage bears no simple relationship to a particular nucleotide order. Our results do not support either the notion that cleavage by RNase E is determined by a consensus sequence or the contrary view that RNase E is a virtually nonspecific single-stranded endonuclease with a preference for cutting 5' to an AU dinucleotide.