Cytoskeleton-bound mRNA for a 40-kDa polypeptide in rat L6 cells is not always translated

The relationship between attachment of mRNA to the cytoskeletal framework and its translation was examined using the mRNA for a polypeptide of 40 kDa (P-40) which is translated in rat L6 myoblasts but not in the myotubes. In both myoblasts and myotubes this mRNA was found to be associated with the cytoskeletal framework. Furthermore, the stability of the association between P-40 mRNA and the cytoskeletal framework in absence of RNA and protein synthesis was examined by using actinomycin D and NaF to block RNA and protein synthesis, respectively. In absence of RNA synthesis portions of both nontranslated P-40 mRNA and translated actin mRNA of myotubes were released into the soluble fraction. In myoblasts, however, both mRNAs remained associated with the cytoskeletal framework following inhibition of RNA synthesis. Inhibition of protein synthesis, on the other hand, had a more dramatic effect on the association between the cytoskeletal framework and P-40 mRNA in myoblasts but not in myotubes. In contrast, the association between actin mRNA and cytoskeletal framework was unaffected by inhibition of protein synthesis in both myoblasts and myotubes. The results of these studies show that the molecular nature of association between cytoskeletal framework and mRNA may differ among mRNAs and may also depend on whether the cells are dividing or are terminally differentiated. Furthermore, no direct relationship between the translation of mRNA and its attachment to the cytoskeletal framework was observed.     

Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA.

The cDNA for mouse thymidine kinase (TK) was isolated from a cDNA library in lambda-gt11 and sequenced. It was used as a probe to follow the time course of TK mRNA expression in growth stimulated mouse fibroblasts. Linked to the HSV-TK promoter the cDNA was able to transform LTK-cells to the TK+ phenotype. The transformed cells expressed the TK mRNA and enzyme activity in a growth dependent fashion suggesting that the regulatory element is localized on the cDNA.     

ERK is regulated by sodium-proton exchanger in rat aortic vascular smooth muscle cells

The purposes of this study were to test 1) the relationship between two widely studied mitogenic effector pathways, and 2) the hypothesis that sodium-proton exchanger type 1 (NHE-1) is a regulator of extracellular signal-regulated protein kinase (ERK) activation in rat aortic smooth muscle (RASM) cells. Angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) stimulated both ERK and NHE-1 activities, with activation of NHE-1 preceding that of ERK. The concentration-response curves for 5-HT and Ang II were superimposable for both processes. Inhibition of NHE-1 with pharmacological agents or by isotonic replacement of sodium in the perfusate with choline or tetramethylammonium greatly attenuated ERK activation by 5-HT or Ang II. Similar maneuvers significantly attenuated 5-HT- or Ang II-mediated activation of MEK and Ras but not transphosphorylation of the epidermal growth factor (EGF) receptor. EGF receptor blockade attenuated ERK activation, but not NHE-1 activation by 5-HT and Ang II, suggesting that the EGF receptor and NHE-1 work in parallel to stimulate ERK activity in RASM cells, converging distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway. Receptor-independent activation of NHE-1 by acute acid loading of RASM cells resulted in the rapid phosphorylation of ERK, which could be blocked by pharmacological inhibitors of NHE-1 or by isotonic replacement of sodium, closely linking the proton transport function of NHE-1 to ERK activation. These studies identify NHE as a new regulator of ERK activity in RASM cells.     

c-myc mRNA is elevated as differentiating lens cells withdraw from the cell cycle

Explants of the central region of lens epithelia from early chicken embryos differentiate in vitro to form lens fiber cells when cultured in the presence of chicken vitreous humor. Hybridization of a 32P-labeled v-myc viral oncogene DNA probe to RNA extracted from differentiating explants and immobilized on nitrocellulose filters indicates that levels of 2.5 kb c-myc mRNA are transiently elevated 5-10-fold in the differentiating cells. Increased levels of c-myc mRNA are observed within 30 min of the initiation of differentiation in vitro and persist for 8-9 h. Thymidine labeling of nuclei in differentiating explants indicates that entry of cells into S phase is inhibited during this period, as differentiating cells complete a final round of mitosis and withdraw from the cell cycle. Levels of c-myc mRNA are also elevated in the peripheral region of the lens epithelium, which contains cells undergoing differentiation in vivo, suggesting that the regulation of c-myc mRNA which occurs in vitro may also occur in vivo. c-myc mRNA, c-fos mRNA, and c-src mRNA showed distinct patterns of regulation associated with lens fiber formation in vivo, thus providing evidence that the regulation of c-myc mRNA is specific to this proto-oncogene. The finding that c-myc mRNA undergoes a specific, transient elevation in differentiating lens cells as they withdraw from the cell cycle contrasts with a large body of evidence linking enhanced c-myc expression with increased cell proliferation.     

Centrioles in the cell cycle of L cells

The structure of centrosome in non-synchronous L-cells culture during the cell cycle has been studied. In mitosis, mother and daughter centrioles, which differ in their ultrastructure, are located perpendicularly in the pole of the spindle. Microtubules, meeting in the pole area terminate mainly in electron-dense clottings of fibrillar matter surrounding the diplosoma. In telophase, disjunction of mother and daughter centrioles begins. At the beginning of G1-period, centrioles move off from each other for several micron, and then draw together again without forming diplosome. Pericentriolar satellites form on mother centriole of some cells at this time, they disappear at the beginning of S-period, replication of centrioles begins; daughter centrioles reach the size of mother centrioles in anaphase. During growth and maturation, centrioles in L-cells undergo structural changes similar to those described for SPEV cells (Vorob'ev, Chentsov, 1982). Several types of meeting points for microtubules exist in L-cells during the whole interphase: surface of centrioles per se, pericentriolar satellites, free foci.     

Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells

We investigated the fate of aberrant mRNAs lacking in-frame termination codons (called nonSTOP mRNA) in mammalian cells. We found that translation of nonSTOP mRNA was considerably repressed although a corresponding reduction of mRNA was not observed. The repression appears to be post-initiation since (i) repressed nonSTOP mRNAs were associated with polysomes, (ii) translation of IRES-initiated and uncapped nonSTOP mRNA were repressed, and (iii) protein production from nonSTOP mRNA associating with polysomes was significantly reduced when used to program an in vitro run-off translation assay. NonSTOP mRNAs distributed into lighter polysome fractions compared to control mRNAs encoding a stop codon, and a significant amount of heterogeneous polypeptides were produced during in vitro translation of nonSTOP RNAs, suggesting premature termination of ribosomes translating nonSTOP mRNA. Moreover, a run-off translation assay using hippuristanol and RNAse protection assays suggested the presence of a ribosome stalled at the 3' end of nonSTOP mRNAs. Taken together, these data indicate that ribosome stalling at the 3' end of nonSTOP mRNAs can block translation by preventing upstream translation events.     

Ganoderma lucidum extract stimulates glucose uptake in L6 rat skeletal muscle cells

The effect of Ganoderma lucidum extract on glucose uptake was studied in L6 rat skeletal muscle cells. G. lucidum extract increased glucose uptake about 2-fold compared to control. The extract stimulated the activity of phosphatidylinositol (PI) 3-kinase which is a major regulatory molecule in the glucose uptake pathway. About 7-fold increased activity of a PI 3-kinase was observed after treatment with G. lucidum extract, whereas PI 3-kinase inhibitor, LY294002, blocked the G. lucidum extract-stimulated PI 3-kinase activity in L6 skeletal muscle cells. Protein kinase B, a downstream mediator of PI 3-kinase, was also activated by G. lucidum extract. We then assessed the activity of AMP-activated protein kinase (AMPK), another regulatory molecule in the glucose uptake pathway. G. lucidum extract increased the phosphorylation level of both AMPK alpha1 and alpha2. Activity of p38 MAPK, a downstream mediator of AMPK, was also increased by G. lucidum extract. Taken together, these results suggest that G. lucidum extract may stimulate glucose uptake, through both PI 3-kinase and AMPK in L6 skeletal muscle cells thereby contributing to glucose homeostasis.     

Syntrophin is an actin-binding protein the cellular localization of which is regulated through cytoskeletal reorganization in skeletal muscle cells

We have characterized the interaction of syntrophin with F-actin. Subcellular fractionation of cardiac and skeletal muscle tissues showed that alpha-, beta1- and beta2-syntrophins were present in the soluble and the membrane fraction. Syntrophins are known to bind to the dystrophin-glycoprotein complex (DGC), but since the DGC is not present in the soluble fraction, it was concluded that some syntrophin did not associate with the DGC. Native syntrophins purified from the soluble fraction and recombinant syntrophins were both able to bind to F-actin, and binding occurred through several sites on syntrophin, including the second pleckstrin homology domain and the unique carboxyl-terminal domain. Syntrophin was also able to inhibit actin-activated myosin ATPase activity and actomyosin super-precipitation. alpha-Syntrophin co-localized with cortical F-actin fibers when expressed in Chinese hamster ovary cells, and deletion of the actin-binding region abolished co-localization. Most of exogenous or endogenous syntrophin also co-localized with stress fibers in endothelial and smooth muscle (A7r5) cells. However, syntrophins were mostly localized in the cytosol of serum-starved C2C12 or primary cultured skeletal muscle myotubes, and translocated to the membrane upon treatment with lysophosphatidic acid or the actin-stabilizing agent jasplakinolide. The actin-depolymerizing agent latrunculin-B abolished this syntrophin translocation. These findings suggest that syntrophin is an actin-binding protein the subcellular localization of which is regulated through cytoskeletal reorganization.     

Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K

Myelination in the central nervous system provides a unique example of how cells establish asymmetry. The myelinating cell, the oligodendrocyte, extends processes to and wraps multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. Local regulation of protein synthesis represents one mechanism used to control the different requirements for myelin sheath at each axo-glia interaction. Prior work has established that β1-integrins are involved in the axoglial interactions that initiate myelination. Here, we show that integrin activation regulates translation of a key sheath protein, myelin basic protein (MBP), by reversing the inhibitory effect of the mRNA 3'UTR. During oligodendrocyte differentiation and myelination α6β1-integrin interacts with hnRNP-K, an mRNA-binding protein, which binds to MBP mRNA and translocates from the nucleus to the myelin sheath. Furthermore, knockdown of hnRNP-K inhibits MBP protein synthesis during myelination. Together, these results identify a novel pathway by which axoglial adhesion molecules coordinate MBP synthesis with myelin sheath formation.     

Cell cycle commitment of rat muscle satellite cells

Satellite cells of adult muscle are quiescent myogenic stem cells that can be induced to enter the cell cycle by an extract of crushed muscle (Bischoff, R. 1986. Dev. Biol. 115:140-147). Here, evidence is presented that the extract acts transiently to commit cells to enter the cell cycle. Satellite cells associated with both live and killed rat myofibers in culture were briefly exposed to muscle extract and the increase in cell number was determined at 48 h in vitro, before the onset of fusion. An 8-12-h exposure to extract with killed, but not live, myofibers was sufficient to produce maximum proliferation of satellite cells. Continuous exposure for over 40 h was needed to sustain proliferation of satellite cells on live myofibers. The role of serum factors was also studied. Neither serum nor muscle extract alone was able to induce proliferation of satellite cells. In the presence of muscle extract, however, satellite cell proliferation was directly proportional to the concentration of serum in the medium. These results suggest that mitogens released from crushed muscle produce long-lasting effects that commit quiescent satellite cells to divide, whereas serum factors are needed to maintain progression through the cell cycle. Contact with a viable myofiber modulates the response of satellite cells to growth factors.