Cancer cells are able to overproduce lactic acid aerobically, whereas normal cells undergo anaerobic glycolysis only when deprived of oxygen. Tumor aerobic glycolysis was recognized about seven decades ago; however, its molecular basis has remained elusive. The lactate dehydrogenase-A gene (LDH-A), whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, was identified as a c-Myc-responsive gene. Stably transfected Rat1a fibroblasts that overexpress LDH-

Expression of the lactate dehydrogenase A subunit (LDH-A) gene can be controlled by transcriptional as well as posttranscriptional mechanisms. In rat C6 glioma cells, LDH-A mRNA is stabilized by activation and synergistic interaction of protein kinases A and C. In the present study, we aimed to identify the sequence domain which determines and regulates mRNA stability/ instability by protein kinase A and focused our attention on the 3-untranslated region (3-UTR) of LDH-A mRNA. We have constructed various chimeric globin/ lactate dehydrogenase (ldh) genes linked to the c-fos promoter and stably transfected them into rat C6 glioma cells. After their transfection, we determined the halflife of transcribed chimeric globin/ldh mRNAs. The results showed that at least three sequence domains within the LDH- Analysis of the LDH 1 isoenzyme patterns in various cell types under a variety of physiologic conditions suggests complex regulatory mechanisms that determine specific isoenzyme expression (1-7). The LDH-A subunit, for instance, is subject to regulation by a number of different effector agents such as estrogen (3,8), epidermal growth factor (5), catecholamines (4, 9), phorbol ester (7), and c-Myc (10), which change the isoenzyme pattern almost exclusively in favor of the LDH-5 (A4) isoenzyme. The functional importance of these LDH isoenzyme shifts is generally attributed to a need for increased A subunitcontaining isoforms (such as LDH-4 or -5), which can derive more energy from the anaerobic pathway by reducing pyruvate to lactate. Investigations into the mechanism of LDH-A gene expression has identified two basic controls consisting of a transcription-regulatory cascade (4, 6, 11) and a mechanism that regulates the half-life of LDH-A mRNA (4, 12), both of which are major determinants of intracellular LDH-A mRNA levels.Messenger RNA turnover rates fluctuate over a wide range, and it is important to identify and characterize putative stability-regulating mRNA domains and their interacting factors that may be responsible for these functional effects. A great number of reports have demonstrated the existence of such domains and their trans-acting regulatory factors that are critical in determining the half-life of mRNA (13). Several of these studies indicate that the stability of some, but not all, mRNA is determined by specific cis-acting AU-rich domains located in the 3Ј-UTR. For example, a number of mRNAs such as cytokine, lymphokine and protooncogene mRNAs share a common sequence motif with a high content of A and U nucleotides in the 3Ј-UTR (14) and exhibit half-lives in the range of only a fraction of 1 h (15-18). In addition, attention has focused on modulation of mRNA stability in response to a variety of physiological signals. For instance, histone mRNA stability is regulated by the cell cycle (19) and intracellular iron levels control the stability of transferrin receptor mRNA (20,21). Moreover, manipulation of cells with several different effector agents can alter the steady-state level of mRNA during cell g...

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.

No abstract

We have identified and studied a posttranscriptional mechanism of lactate dehydrogenase A (LDH) subunit gene expression at the level of mRNA stability. Using the well differentiated rat C6 glioma cell line as a model system, the effects of activators of the protein kinase A and C pathways on the half-life of LDH A mRNA were measured by two independent methods: 1) by the RNA synthesis inhibitor-chase method using actinomycin D, and 2) by analysis of decay of LDH A [3H]mRNA in [3H]uridine-labeled cells. By each method, the half-life of relatively short-lived LDH A mRNA was increased 5- to 7-fold in 8- (4-chloro-phenylthio) cAMP or forskolin-treated and about 3-fold in 12-0-tetradecanoylphorbol-13- acetate (TPA) or dioctanoylglycerol-treated cells. Forskolin acted synergistically with TPA to prolong LDH A mRNA half-life from 55 min to more than 20 h. The relatively rapid basal decay rate of LDH A mRNA was also considerably slowed in the presence of the protein phosphatase inhibitor okadaic acid, suggesting a functional role for protein phosphorylation in the stabilization process. In glioma cells stably transformed with a protein kinase A catalytic subunit expression vector, overexpression of the catalytic subunit stabilized LDH mRNA to the degree seen in forskolin-treated cells. In cells transfected with a protein kinase A inhibitor-expression vector, cAMP-mediated stabilization of LDH A mRNA half-life was prevented. Furthermore, both staurosporin and 3- [1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol- 3-yl)- maleimide, inhibitors of protein kinase C, prevented the TPA-induced stabilization of LDH A mRNA. We conclude from the experimental data that the protein kinase A and C signal pathways play an active functional role in regulating LDH A mRNA stability and act cooperatively to achieve LDH A mRNA stability regulation.

We have explored the molecular basis of the cAMPinduced stabilization of lactate dehydrogenase A (LDH-A) mRNA and identified four cytoplasmic proteins of 96, 67, 52, and 50 kDa that specifically bind to a 30-nucleotide uridine-rich sequence in the LDH 3-untranslated region with a predicted stem-loop structure. Mutational analysis revealed that specific protein binding is dependent upon an intact primary nucleotide sequence in the loop as well as integrity of the adjoining double-stranded stem structure, thus indicating a high degree of primary and secondary structure specificity. The critical stem-loop region is located between nucleotides 1473 and 1502 relative to the mRNA cap site and contains a previously identified cAMP-stabilizing region (CSR) required for LDH-A mRNA stability regulation by the protein kinase A pathway. The 3-untranslated region binding activity of the proteins is upregulated after protein kinase A activation, whereas protein dephosphorylation is associated with a loss of binding activity. These results imply a cause and effect relationship between LDH-A mRNA stabilization and CSR-phosphoprotein binding activity. We propose that the U-rich CSR is a recognition signal for CSR-binding proteins and for an mRNA processing pathway that specifically stabilizes LDH mRNA in response to activation of the protein kinase A signal transduction pathway.There is increasing evidence demonstrating that the rate of mRNA turnover and its regulation by effector agents play significant roles in controlling mRNA steady-state levels (1, 2). We have previously demonstrated, for example, that not only does the relatively high steady-state level of LDH-A 1 mRNA after ␤-adrenergic agonist or phorbol ester stimulation of rat C6 glioma cells reflect an increased transcriptional rate (3, 4) but that cells also have the ability to enhance the intracellular mRNA level by a mechanism that regulates the rate of degradation and half-life of mRNA through activation of protein kinases A and C (5). Selective mRNA stabilization in eukaryotic cells by stimulants of second messenger pathways, although not as widely and thoroughly studied as transcriptional control, is a regulated property that can determine the level of expression of a gene product. It is known that individual mRNAs within eukaryotic cells can display a wide range of stability, with half-lives ranging from a few minutes for highly regulated gene products, such as oncogenes, to over 24 h for very stable species, such as ␤-globin mRNA (1, 6 -8).Lactate dehydrogenase A subunit mRNA is characterized by a relatively short half-life of about 45-55 min (4, 5). Agents that activate protein kinases A or C cause a 9-fold or 4-fold increase, respectively, in the half-life of LDH-A mRNA (4, 5). Recent analysis of the factors regulating stability of LDH-A mRNA has led to the recognition of a cAMP-stabilizing region (CSR), the presence of which in the 3Ј-UTR is absolutely necessary for cAMP regulation of LDH mRNA stability to occur (9). In the present report, we describe the e...

No abstract

No abstract