Expression of the Gs protein alpha-subunit disrupts the normal program of differentiation in cultured murine myogenic cells.

The manner in which growth factors acting at the cell surface regulate activity of myogenic basic-helix-loop-helix proteins in the nucleus and thus control the fate of committed skeletal myoblasts remains poorly understood. In this study, we report that immunoreactive Gs protein alpha-subunits (Gs alpha) localize to nuclei of proliferating C2C12 myoblasts but not to nuclei of differentiated postmitotic C2C12 myotubes. To explore the biological significance of this observation, we placed a cDNA encoding Gs alpha in an expression vector under the control of a steroid-inducible promoter and isolated colonies of stably transfected C2C12 myoblasts. Dexamethasone-induced expression of activated Gs alpha markedly delayed differentiation in comparison with uninduced stably transfected cells, which differentiated normally in mitogen-depleted media. Northern blot analysis showed that impaired differentiation was associated with delayed up-regulation of MyoD and myogenin and delayed down-regulation of Id, a dominant negative inhibitor of differentiation. Similar impairment of differentiation could not be reproduced in wild-type C2C12 cells by increasing intracellular cAMP either with forskolin or treatment with a cell-permeable cAMP analog. However, treatment of myoblasts with cholera toxin markedly inhibited myogenic differentiation. Taken together, these findings suggest a novel role for Gs alpha in modulating myogenic differentiation.

Characterization of MB creatine kinase isoform conversion in vitro and in vivo in dogs.

Time-dependent removal of the COOH-terminal lysine residue from each subunit of tissue MM creatine kinase by plasma carboxypeptidase N produces two additional isoforms that are readily separated, thereby permitting sensitive, early detection of acute myocardial infarction. Only two isoforms of MB creatine kinase have been detected in plasma leading to speculation that the COOH-terminal lysine on the B subunit is resistant to hydrolysis. To define the biochemical changes resulting in MB creatine kinase isoform conversion, we incubated highly purified MB creatine kinase from canine myocardium with plasma carboxypeptidase N. Quantitative anion-exchange chromatography of incubation mixtures and serial plasma samples from dogs subjected to coronary occlusion revealed a second, more acidic form evolved with time that was separated from the tissue isoform. Cyanogen bromide digestion of the two isoforms followed by amino acid sequencing of COOH-terminal peptides showed that MB creatine kinase undergoes removal of the COOH-terminal lysine residue from both M and B subunits. An intermediate form lacking lysine on the M subunit was delineated during incubations by the combined use of anion-exchange chromatography and conventional electrophoretic techniques. Thus, sequential cleavage of lysine from subunits of MB creatine kinase produces an intermediate isoform that has not been detected previously because of difficulties separating it from the tissue and fully converted isoforms.

Tissue-specific distribution and developmental regulation of M and B creatine kinase mRNAs.

To characterize the tissue-specific distribution and developmentally regulated expression of M and B creatine kinase mRNA in rats, total cellular RNA was isolated from adult rat tissues and from skeletal muscle, heart, brain and intestine at selected stages of development. Northern blots were prepared and hybridized with M and B subunit-specific probes derived from the 3'-untranslated region. M creatine kinase mRNA was expressed abundantly in heart and skeletal muscle, and less abundantly in lung. B creatine kinase mRNA was found in all tissues examined except liver and was abundant in brain, heart and intestine. The developmentally regulated expression of M and B creatine kinase mRNA was determined in skeletal muscle, heart, brain and intestine. The developmental program of B creatine kinase mRNA was different for each tissue examined. During development, M creatine kinase mRNA was up-regulated in both heart and skeletal muscle with a different regulatory program. This resulted in replacement of B mRNA by M mRNA as the predominant species at an earlier developmental stage in heart when compared to skeletal muscle.

Identification and localization of a fatty acid response region in the human plasminogen activator inhibitor-1 gene.

The increased expression of plasminogen activator inhibitor type-1 (PAI-1) is associated with increased concentrations of fatty acids in blood and may accelerate atherogenesis in diabetes. The present study was designed to define mechanisms by which nonesterified (free) fatty acids (FFAs) augment the expression of PAI-1. FFAs increased PAI-1 protein and mRNA expression by HepG2 cells. To identify potential regulatory elements, we constructed chimeric genes by fusing 1313, 853, 610, or 328 bp of human PAI-1 5'-flanking DNA to a luciferase reporter (PAI-LUC). A 2-fold increase in luciferase activity was seen when cells were transfected with PAI-LUC 1313, 863, or 610 and exposed to FFAs. No response to FFAs was seen with PAI-LUC 328 and after deletion of a 72-bp (-599 to -528) fragment from PAI-LUC 1313. This 72-bp fragment conferred FFA responsiveness to a different (simian virus 40) promoter. Two footprinted regions were demonstrated by DNase I analysis. Gel mobility shift assays indicated specific binding of extracted proteins to an FFA response element: 5'-TG(G/C)(1-2)CTG-3'. This sequence is repeated 4 times and is similar to an Sp1-binding site. Sp1 consensus oligonucleotides inhibited binding of extracted proteins to the regulatory elements. Accordingly, FFA-induced increased expression of PAI-1 in HepG2 cells is mediated by the binding of a transcription factor or factors to the repeated fatty acid response element, 5'-TG(G/C)(1-2)CTG-3', that is highly homologous to an Sp1-binding site.

Labeling of human clots in vitro with an active-site mutant of t-PA.

Prompt detection of acute thrombosis and its response to treatment with thrombolytic agents generally require angiography. Scintigraphic approaches with labeled antibodies to or components of the coagulation and fibrinolytic systems have been disappointing because of prolonged circulating half-lives of tracers and relatively slow or limited binding to thrombi. Accordingly, we developed and characterized a thrombolytically inactive, active-site mutant (Ser-478----Thr) of tissue-type plasminogen activator (t-PA) designed to detect thrombi in vivo. Binding of iodine-125-(125I) labeled Ser----Thr t-PA to thrombi in vitro was time- and concentration-dependent, and specific judging from inhibition by pre-incubation with anti-t-PA IgG. Clearance of 125I-labeled mutant t-PA in rabbits was rapid and biexponential (alpha t1/2 = 1.9 +/- 0.4 min, beta t1/2 = 39.8 +/- 11.2 min). Thus, the amidolytically inactive mutant of t-PA designed binds rapidly and specifically to human thrombi in vitro and is cleared rapidly from the circulation in vivo--properties rendering it attractive as a potentially useful clot imaging agent.

Mediators of induction of augmented expression of plasminogen activator inhibitor type-1 in Hep G2 cells by platelets.

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. We have shown previously that PAI-1 biosynthesis in cultured cells depends on several factors in serum. Because platelets are richly endowed with specific growth factors and because the release reaction is an integral part of thrombosis, the present study was performed to determine whether platelets augment PAI-1 production and if so, to define mediators responsible. Hep G2 cells were used to determine whether platelet lysates increased PAI-1 synthesis in a dose and time-dependent manner. In cells labeled metabolically with 35S-methionine for 6 h, an increase in labeled PAI-1 was elicited indicative of de novo synthesis as well as increased secretion of PAI-1 mediated by platelet lysates. Steady state levels of both the 3.2 and 2.2 kb forms of PAI-1 mRNA increased after 2 h and peaked in 3-5 h in a dose-dependent fashion as well. Incubation of Hep G2 cells with collagen activated platelets resulted in a similar induction of PAI-1 mRNA. The increase in PAI-1 mRNA occurred with exposure of the cells to platelet lysates for intervals as brief as 15 min and was not inhibited by cycloheximide indicating its independence of new protein synthesis. In order to identify the factors in platelets responsible for the induction of PAI-1 synthesis in the Hep G2 cell model system, neutralizing antibodies were used to inhibit specific platelet associated growth factors. Antibodies to transforming growth factor-beta (TGF-beta) and to the epidermal growth factor (EGF)/transforming growth factor alpha (TGF-alpha) receptor inhibited the platelet lysate-mediated increase in PAI-1 protein by 77%.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of synthesis of plasminogen activator inhibitor type-1 by tissue-type plasminogen activator in human hepatic and endothelial cells.

Plasminogen activator inhibitor type-1 (PAI-1) can modify fibrinolytic activity in vitro and in vivo. The present study was performed to determine whether pharmacologic concentrations of tissue-type plasminogen activator (t-PA) can initiate negative feedback by stimulating PAI-1 synthesis. In both human hepatoma cells (Hep G2) and human umbilical vein endothelial cells (HUVEC), t-PA increased the total concentrations and appearance of newly synthesized protein in conditioned media of free PAI-1 and PAI-1 complexed with t-PA in a dose and time dependent fashion judging from results after immunoprecipitation of metabolically labeled PAI-1. The t-PA effect was not attributable simply to release of stored or matrix-bound PAI-1. In HUVEC, Northern blot analyses indicated that t-PA increased steady-state levels of PAI-1 mRNA two-fold. In contrast PAI-1 mRNA expression was not increased in Hep G2 cells. Thus, mechanisms of stimulation appeared to differ in the two cell lines. The results obtained are consistent with the hypothesis that increased PAI-1 synthesis and secretion in response to t-PA may limit or attenuate fibrinolysis locally or systemically in vivo.

Potential attenuation of fibrinolysis by growth factors released from platelets and their pharmacologic implications.

Increased concentrations of the fast-acting tissue-type plasminogen activator (t-PA) inhibitor attenuate the fibrinolytic activity of pharmacologically administered activators of the fibrinolytic system such as t-PA. Accordingly, it was hypothesized that augmentation of synthesis and elaboration of inhibitor from the liver, leading to increased concentrations of inhibitor in plasma, or from endothelial cells in the vicinity of thrombi undergoing lysis, leading to increased concentrations locally, may contribute to failure of pharmacologically induced thrombolysis or to early reocclusion. Because platelets are rich in transforming growth factor beta and epidermal growth factor-like activity, it was thought that release of growth factors from platelets activated in vivo could mediate increases of the inhibitor in plasma by stimulating its formation in the liver and its local release from endothelial cells in the vicinity of thrombi. If so, fibrinolysis might be rendered more effective by concomitant prevention of platelet growth factor release. Transforming growth factor beta, a major constituent of platelets, increased concentrations of the t-PA inhibitor messenger ribonucleic acid (mRNA) in human hepatoma cells in a specific and dose-dependent manner. A peak effect was seen with 5 ng/ml and a 10-fold increase in 6 hours. Release of inhibitor protein into conditioned media increased as well. Induction of the inhibitor mRNA increase was elicited by exposure as brief as 30 minutes. Cycloheximide, an inhibitor of protein synthesis, was not inhibitory. The mechanisms responsible differed from those seen with epidermal growth factor, shown previously in the laboratory to increase inhibitor mRNA. In addition, the 2 factors were synergistic. Platelet lysates elicited effects simulating those of the purified growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of creatine kinase M and B mRNAs in treadmill trained rat skeletal muscle.

Gastrocnemius muscle from treadmill trained rats was analyzed for creatine kinase (CK) isoenzyme activities by agarose electrophoresis and M and B CK mRNA levels by Northern blot analysis. Total CK activity in exercise-trained (143 (SD 15) U/g) and control (154 (SD 16) U/g) muscles did not differ. CK-MB increased 220% in exercised-trained muscle compared to controls. CK-B subunit mRNA increased 40%, while CK-M subunit mRNA decreased 42% in exercised-trained muscle compared to control. Thus, gene expression of CK isoenzymes appears to be partially controlled at the level of transcription following exercise training.

Species-specific differential cleavage and polyadenylation of plasminogen activator inhibitor type 1 hnRNA.

Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiologic inhibitor of the naturally occurring plasminogen activators. In higher primates two forms of mature PAI-1 mRNA (3.2 kb and 2.2 kb) arise by alternative cleavage and polyadenylation of PAI-1 hnRNA which is regulated in a tissue-specific fashion in humans. In other mammals only the 3.2 kb mRNA has been detected. The putative downstream polyadenylation site in humans that gives rise to the 3.2 kb PAI-1 mRNA consists of three overlapping copies of the consensus polyadenylation sequence while no consensus polyadenylation sequence is found upstream at a position that could generate the shorter mRNA species. To determine whether differential cleavage and polyadenylation of PAI-1 mRNA is due to species-specific differences in trans-acting factors that process PAI-1 mRNA or to the presence of a nonconsensus polyadenylation site acquired recently during primate evolution we prepared plasmids in which the 3' nontranslated region of the human PAI-1 gene or the mouse PAI-1 cDNA was inserted downstream of the neomycin gene in the plasmid pSV2neo. We show that the 3'-nontranslated region of the human PAI-1 gene but not the mouse PAI-1 cDNA conferred alternative cleavage and polyadenylation to the neomycin gene in transfected human Hep G2 cells as well as mouse NIH3T3 and rat L6 cells.

Characterization of a nuclear protein that interacts with regulatory elements in the human B creatine kinase gene.

The B creatine kinase gene is regulated by an array of positive and negative cis-elements in the 5'-flanking DNA that function in both muscle and nonmuscle cells. In C2C12 myogenic cells M and B creatine kinase mRNAs are coordinately up-regulated in the early stages of myogenesis and then undergo distinct regulatory programs. The B creatine kinase gene is down-regulated in the late stages of myogenesis as M creatine kinase becomes the predominant species in mature myotubes. Sequences between -92 and +80 of the B creatine kinase gene confer a regulated pattern of expression to chimeric plasmids that closely resembles the time-course of expression of the endogenous B creatine kinase gene in C2C12 cells undergoing differentiation. We show that sequences within the first exon of the B creatine kinase gene are important for the development regulation of the gene in C2C12 cells and that these sequences bind a nuclear protein that shows a similar tissue-specific distribution and developmentally regulated expression to that of the endogenous B creatine kinase gene.

Multiple transforming growth factor-beta-inducible elements regulate expression of the plasminogen activator inhibitor type-1 gene in Hep G2 cells.

Regulation of plasminogen activation by plasminogen activator inhibitor type-1 (PAI-1) is a critical feature of many biological processes. Transforming growth factor-beta (TGF-beta) induces PAI-1 mRNA and protein in several types of cultured cells, including Hep G2 cells. The present study was performed to define mechanisms by which PAI-1 gene expression is regulated by TGF-beta. Nuclear run-on assays performed on Hep G2 cells stimulated with TGF-beta for 6 h showed a 3.8-fold increase in PAI-1 gene transcription. TGF-beta increased the half-life of PAI-1 mRNA in Hep G2 cells 2.5-fold over control values. To characterize transcriptional regulatory mechanisms, we constructed chimeric genes containing PAI-1 5'-flanking DNA fused upstream of the bacterial chloramphenicol acetyltransferase gene in the vector pSVOCAT and transfected Hep G2 cells. Promoter deletion analysis demonstrated that sequences between -791 and -328 and -328 to -186 base pairs upstream of the PAI-1 gene cap site contain TGF-beta responsive elements that conferred TGF-beta inducibility in an orientation and position-independent manner. Further characterization of the larger TGF-beta-inducible enhancer (-791 to -328) located a TGF-beta-inducible element at nucleotides -791 to -546 upstream of the PAI-1 gene cap site. These results demonstrate that PAI-1 gene regulation by TGF-beta in Hep G2 cells is mediated both at a transcriptional level by two specific inducible elements, as well as by post-transcriptional mechanisms.

Developmental regulation and tissue-specific expression of the human muscle creatine kinase gene.

To define mechanisms regulating expression of M creatine kinase, the human gene including 5'-flanking DNA was cloned, characterized, and partially sequenced. The gene contains 8 exons interrupted by 7 introns spanning 17.5 kilobase pairs of DNA. The intron-exon splice sites were identified and conform to the GT-AG consensus rule. The TATA and CAAT boxes are located at positions -31 and -56 upstream of the transcription start site as determined by primer extension. The 5'-untranslated region is interrupted with the translation start codon located in the second exon. To determine whether sequences within the 5'-upstream DNA confer tissue-specific expression and developmental regulation, constructs containing 2620 base pairs of human M creatine kinase 5'-flanking DNA fused upstream of the chloramphenicol acetyltransferase gene in the promoterless plasmid pSVO-CAT were transfected into cultured C2C12 myoblasts. There was 17-fold induction of chloramphenicol acetyltransferase activity during differentiation as C2C12 myoblasts fused to form myotubes. The M creatine kinase fusion construct was not expressed in transfected nonmuscle cell lines, COS-7 and NIH/3T3. Thus, cis-acting sequences within 2620 base pairs of the cap site are sufficient to direct developmental regulation and tissue-specific expression of the human M creatine kinase gene.

Regulation of the expression of type 1 plasminogen activator inhibitor in Hep G2 cells by epidermal growth factor.

To identify factors potentially influencing expression of type 1 plasminogen activator inhibitor (PAI-1), we characterized the human tissue-specific distribution of PAI-1 mRNA and the influence of epidermal growth factor (EGF) on expression of steady state levels of PAI-1 mRNA and secretion of PAI-1 by Hep G2 cells. Two species of PAI-1 mRNA (3.2 and 2.2 kilobases) were detected, and the ratio of the two varied among tissues (3 to 5:1) in contrast to the 1:1 ratio detected in Hep G2 cells. Expression of PAI-1 mRNA was inversely related to the distribution of tissue-type plasminogen activator mRNA (2.3 kilobases). Nu-Serum, a growth media supplement, increased steady state levels of PAI-1 mRNA 5-fold within 3 h. Factors responsible were found to be trypsin-sensitive and dialysis-resistant. Antisera to EGF attenuated Nu-Serum-induced increases of PAI-1 mRNA by 57%, suggesting that EGF or EGF homologous peptides contributed to the response. EGF elicited increases of PAI-1 mRNA levels in a dose-dependent manner. Induction was rapid (7-fold at 3 h with 5 ng/ml) and complete within 10 h. The response was not attenuated by cycloheximide (25 micrograms/ml). Factor X and glyceraldehyde-3-phosphate dehydrogenase mRNA did not increase. Increased levels of PAI-1 antigen were detected in conditioned media of Hep G2 cells by 4 h and were maximal at 8 h (6-fold). We conclude that the expression of PAI-1 mRNA is tissue-specific and regulated by epidermal growth factor in Hep G2 cells.

Determination of intact-tissue glycerophosphorylcholine levels by quantitative 31P nuclear magnetic resonance spectroscopy and correlation with spectrophotometric quantification.

A method for the dynamic quantification of glycerophosphorylcholine (GPC) levels in intact tissue by 31P nuclear magnetic resonance spectroscopy was developed and verified by a spectrophotometric technique. Intact tissue nuclear magnetic resonance areas were quantified utilizing an external standard and were corrected for magnetization saturation. Interactive computerized spectral fitting through Lorentzian lineshape analysis and subsequent integration with normalization to the external standard was utilized for the absolute quantification of GPC concentration. Hemodynamically and metabolically uncompromised Langendorff perfused rabbit hearts contained 1.70 +/- 0.23 mumol GPC/g wet wt. This value was not statistically significantly different from the value of 1.45 +/- 0.23 mumol GPC/g wet wt determined by an analytical technique employing glycerophosphoryl-[Me-3H]choline as an internal standard with spectrophotometric quantification. Both methods were accurate with a standard error of 11 and 10%, respectively. The recovery of internal standards utilizing the spectrophotometric technique was 95 +/- 8%. The application of these methods should facilitate the quantification of changes in tissue levels of glycerophosphorylcholine noted in several disease states.

The complete nucleotide sequence of canine brain B creatine kinase mRNA: homology in the coding and 3' noncoding regions among species.

To define the structure of canine B creatine kinase, clones were isolated from a library prepared from dog brain mRNA and constructed in the vector lambda gt11. The entire coding portion, the complete 3' nontranslated region, and 43 bp of the 5' noncoding region are reported. Comparison of the predicted amino acid sequence of canine B creatine kinase with the sequence of canine M creatine kinase shows 81% identity. When compared to cDNAS encoding B creatine kinase isolated form other species unusual and striking nucleotide sequence identity in the 3' noncoding region is present. Moreover, two B creatine kinase clones (BCK2 and BCK38) demonstrate microheterogeneity within the 3' nontranslated region indicating variable processing of B creatine kinase pre-mRNA or the existence of multiple genes encoding canine B creatine kinase.

A novel protein interacts with the major transforming growth factor-beta responsive element in the plasminogen activator inhibitor type-1 gene.

Multiple transforming growth factor-beta (TGF-beta) responsive elements have been identified within the 5'-flanking region of the plasminogen activator inhibitor type-1 (PAI-1) gene. This study was designed to characterize the major TGF-beta responsive element (-804 to -546). DNA footprint assays showed that the region of protein contact (-726 to -703) did not include consensus sequences for any known transacting factors. The results of UV cross-linking and Southwestern blot experiments showed that a protein of M(r) 100,000 specifically binds to the TGF-beta responsive element and that this protein undergoes post-transcriptional activation within 5 min after stimulation of Hep G2 cells by TGF-beta resulting in a marked increase in affinity for the target DNA sequence. These results show that stimulation of Hep G2 cells with TGF-beta increases the affinity of a novel 100-kDa protein for the major TGF-beta responsive element within the PAI-1 gene.

Transcriptional regulation of plasminogen activator inhibitor type-1 mRNA in Hep G2 cells by epidermal growth factor.

Secretion of plasminogen activator inhibitor type-1 (PAI-1) by cultured cells is increased after exposure to specific cytokines and growth factors. We have shown previously that incubation of Hep G2 cells with epidermal growth factor (EGF) results in a marked increase in steady state levels of PAI-1 mRNA (Lucore, C.L., et al. (1988) J. Biol. Chem. 263, 15845-15848). The present study was undertaken to determine whether the regulation of expression of PAI-1 mRNA by EGF is mediated at the level of transcription and/or by post-transcriptional mechanisms. The rate of transcription of the PAI-1 gene measured by nuclear run-on assays was found to be increased within 2 h after stimulation of the cells with EGF (5 ng/ml) (3.2 fold increase relative to control, n = 2, range 3.0-3.4). It reached a maximum in 3 h, (9.2 fold increase relative to control, n = 2, range 8.8-9.6) and returned to baseline in 5 h. Exposure of the cells to EGF did not increase the rate of transcription of the glyceraldehyde-3-phosphate dehydrogenase gene. The half life of PAI-1 mRNA in Hep G2 cells was 120 min as determined by RNA blot analysis after exposure of the cells to actinomycin D to inhibit transcription. Stimulation of the cells with EGF did not result in significant change in the half life of PAI-1 mRNA. The results demonstrate that exposure of Hep G2 cells to EGF increases PAI-1 gene transcription.