A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo.

The tumor necrosis factor alpha (TNF-alpha) gene is rapidly activated by lipopolysaccharide (LPS). Here, we show that extracellular signal-regulated kinase (ERK) kinase activity but not calcineurin phosphatase activity is required for LPS-stimulated TNF-alpha gene expression. In LPS-stimulated macrophages, the ERK substrates Ets and Elk-1 bind to the TNF-alpha promoter in vivo. Strikingly, Ets and Elk-1 bind to two TNF-alpha nuclear factor of activated T cells (NFAT)-binding sites, which are required for calcineurin and NFAT-dependent TNF-alpha gene expression in lymphocytes. The transcription factors ATF-2, c-jun, Egr-1, and Sp1 are also inducibly recruited to the TNF-alpha promoter in vivo, and the binding sites for each of these activators are required for LPS-stimulated TNF-alpha gene expression. Furthermore, assembly of the LPS-stimulated TNF-alpha enhancer complex is dependent upon the coactivator proteins CREB binding protein and p300. The finding that a distinct set of transcription factors associates with a fixed set of binding sites on the TNF-alpha promoter in response to LPS stimulation lends new insights into the mechanisms by which complex patterns of gene regulation are achieved.

Molecular characterization of human brain N-acetylated alpha-linked acidic dipeptidase (NAALADase).

N-Acetylated alpha-linked acidic dipeptidase (NAALADase) is a neuropeptidase that may modulate glutamatergic neurotransmission. Independent of its characterization in the nervous system, one form of NAALADase was shown to be expressed at high levels in human prostatic adenocarcinomas, and it was designated the prostate-specific membrane antigen (PSMA). The NAALADase/PSMA gene is known to produce multiple mRNA splice forms, and based on previous immunohistochemical evidence, it had been assumed that the human brain and prostate expressed different isoforms of the enzyme. Because PSMA is being actively pursued as a target for autoimmune and cytotoxic targeting strategies to treat prostate cancer, the rigorous comparison of the two forms of the enzyme remained an important but untested question. To assess similarities and/or differences between human brain NAALADase and PSMA, we compared the two molecules using criteria of activity, immunoreactivity and sequences of the corresponding mRNAs. NAALADase from human cerebellar isolates displayed a kinetic profile and pharmacological sensitivities similar to PSMA. Also, Northern hybridization to PSMA cDNA detected indistinguishable sets of 2.8-, 4.0- and 6.0-kb RNA species in human brain and the LNCaP prostatic tumor cell line. In addition, the monoclonal antibody 7E11-C5 directed against the prostatic form of the enzyme immunoprecipitated 82% of human cerebellar NAALADase activity. Moreover, reverse transcription-polymerase chain reaction cloning of cerebellar cDNAs indicated that the human brain and prostate express a common mRNA splice form. Therefore, we conclude that the form of NAALADase also known as PSMA is expressed in brain and comprises a significant fraction of brain NAALADase activity.

Hydrolysis of the neuropeptide N-acetylaspartylglutamate (NAAG) by cloned human glutamate carboxypeptidase II.

Glutamate carboxypeptidase II may modulate excitatory neurotransmission through the catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) and possibly other endogenous peptide substrates. To investigate the molecular properties of cloned human GCP II (hGCP II), we analyzed the NAAG-hydrolytic activity conveyed by transfection of a full-length hGCP II cDNA into PC3 cells, which do not express GCP II endogenously. Membrane fractions from these cells demonstrated activity with an apparent Km of 73 nM and Vmax of 35 pmol/(mg protein*min). Activity was inhibited by EDTA and stimulated by the addition of CoCl2. Addition of GCP II inhibitors beta-NAAG, quisqualic acid and 2-(phosphonomethyl)pentanedioic acid (PMPA) inhibited hydrolysis of 2.5 nM NAAG with IC50s of 201 nM, 155 nM and 98 pM, respectively. In competition experiments designed to infer aspects of hGCP II substrate selectivity, NAAG was the most potent alpha peptide tested, with an IC50 of 26 nM. Folate derivatives and some other gamma-glutamyl peptides showed comparable affinity to that of NAAG, also displaying IC50s in the low nM range. Taken together with previous evidence demonstrating their presence in GCP II-expressing tissues, these data suggest that both NAAG and folates are good candidate substrates for GCP II in vivo.

Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II.

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length. In situ hybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.