Kamal D. Mehta

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38MAPK, induces low density lipoprotein (LDL) receptor expression (6–8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38MAPK signaling pathway by expression of MKK6b(E), a constitutive activator of p38MAPK, significantly reduced LDL receptor promoter activity. Expression of the p38MAPK α-isoform had a similar effect, whereas expression of the p38MAPK βII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44MAPK, and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38MAPK negatively regulates the p42/44MAPK cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44MAPK activity did not affect p38MAPK activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38MAPK and p42/44MAPK and provide the first evidence that through the p42/44MAPK signaling cascade, the p38MAPKα-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.

Identification of a Novel cis-Acting Element Participating in Maximal Induction of the Human Low Density Lipoprotein Receptor Gene Transcription in Response to Low Cellular Cholesterol Levels

In this paper, we present both in vivo and in vitro evidence for the presence of a novel cis-acting regulatory element that is required for maximal induction of the human low density lipoprotein (LDL) receptor gene following depletion of cellular sterols in HepG2 cells. First, in vivo dimethyl sulfate footprinting of the human LDL receptor promoter before and after transcriptional induction in HepG2 cells revealed protection from −145 to −126, 5′-GAGCTTCACGGGTTAAAAAG-3′ (referred to as FP1 site). Second, transient transfections of HepG2 cells with promoter luciferase reporter constructs containing the FP1 site resulted in significant enhancement (approximately 375%) of reporter gene expression in response to low levels of sterols compared with parallel plasmid without the FP1 site. In addition, this response was markedly attenuated on nucleotide substitutions within the FP1 site. Third, by electrophoretic mobility shift assays, the FP1 sequence was found to bind protein(s) from HepG2 nuclear extracts in a sequence-specific manner. In vitro binding of the FP1 mutants paralleled the results obtained for their in vivo transcription. On the basis of competition profiles, the FP1-binding factor is different from the known transcription factors binding to the AT-rich CArG and GArC motifs. Furthermore, the FP1-binding protein is not specific to HepG2 cells because nuclear factor(s) with the same specificity was observed in nuclear extracts of non-hepatic HeLa cells. We conclude that transcriptional induction of the LDL receptor gene in response to sterol depletion is mediated, in part, by an highly conserved novel cis-acting element through the binding of specific nuclear protein(s).

Inhibition of Stress-Activated p38 Mitogen-Activated Protein Kinase Induces Low-Density Lipoprotein Receptor Expression

We have recently shown that different signal transduction pathways initiated by a variety of agents converge on growth-responsive p42/44MAPK signaling cascade to induce low-density lipoprotein (LDL) receptor expression. Our recent demonstration that stress-activated p38MAPK negatively regulates LDL receptor expression in an isoform-specific manner via modulation of p42/44MAPK cascade represents a new dimension of complexity in the molecular communication that governs LDL receptor expression. The suggested one-way communication between p38MAPK and p42/44MAPK provides a potential mechanism for fine-tuning cellular levels of cholesterol in response to a diverse set of environmental cues, including stress. Cross talk between MAPKs opens new avenues toward understanding a variety of pathogenic processes; this makes them tempting targets for therapeutic interventions in cardiovascular diseases.

Genetic complexity of the human geranylgeranyltransferase I β-subunit gene: a multigene family of pseudogenes derived from mis-spliced transcripts

Geranylgeranyltransferase I controls the function of a variety of cellular proteins by attaching a geranylgeranyl group to the carboxy-terminus of proteins. The purified enzyme from rat brain is comprised of two polypeptides, a catalytic alpha-subunit (GGTalpha) and a substrate-binding beta-subunit (GGTbeta). The present paper demonstrates the existence of a GGTbeta multigene family in humans by describing the presence and characterization of at least 13 pseudogenes related to this protein. Sequencing of numerous PCR-derived clones, obtained following amplification of human genomic DNA, revealed multiple, distinct but highly related sequences. All clones had a common deletion of 99-bp that conforms to the GT-AG rule of splicing in eukaryotes, and differed from the human GGTbeta cDNA sequence by multiple nucleotide substitutions. PCR amplification from mRNA, however, yielded only the sequence expected for the expressed GGTbeta protein. This apparent paradox was resolved by cloning and sequencing a complete GGTbeta-specific pseudogene. Multiple features of the cloned gene, in particular the absence of introns, presence of flanking direct repeats, and the lack of sequence similarity with the untranscribed region of the gene, indicate that this clone represents a processed pseudogene possibly resulting from a mis-spliced transcript. Multiple GGTbeta-specific pseudogenes appear to have resulted from more than one retroposition event. These results suggest a potential role for mis-splicing in the evolutionary diversity of pseudogenes.

p21ras farnesyltransferase α- and β-subunits are phosphorylated in PC-12 cells: TGF-β signaling pathway independent phosphorylation

Farnesyltransferase (FTase) catalyzes the transfer of a farnesyl isoprenoid to the conserved carboxyl-terminal cysteine residue of proteins terminating with the CAAX sequence. Rat brain FTase is a heterodimer consisting of a 49 kDa α-subunit and a 46 kDa β-subunit. In this report, we show, for the first time, that the β-subunit of FTase is phosphorylated in vivo and the FTase heterodimer contains phosphorylated α/β-subunits in rat adrenal medulla pheocytochroma PC-12 cells. The presence of the phosphorylated FTase subunits as heterodimer in PC-12 cells which are known to be deficient in TGF-β signaling pathways argues against the involvement of this pathway in their phosphorylation and heterodimerization.

CHILOSCYLLIUM PLAGIOSUM LOW-DENSITY LIPOPROTEIN RECEPTOR : EVOLUTIONARY CONSERVATION OF FIVE DIFFERENT FUNCTIONAL DOMAINS

All five functional domains of the low-density lipoprotein (LDL) receptor were assembled in their modern form more than 450 million years ago, as revealed from the cloning and sequencing of an LDL receptor cDNA fromChiloscyllium plagiosum (banded cat shark). The shark LDL receptor has the same overall architecture as the mammalian and amphibian counterparts. Each of the seven cysteine-rich repeats in the ligand binding domain resembles its counterpart in the human LDL receptor more than it does the other repeats in the shark receptor as suggested by the presence of unique “signature” sequences, indicating that these repeats had already acquired their independent structures by the time of shark development. Furthermore, amino acid sequences of the entire ligand binding domain of shark LDL receptor show 35% identity over a stretch of 294 residues with aLymnaea stagnalis G-protein-linked receptor (LSGLR). The region of homology between these unrelated proteins includes conservation of most of the unique characteristics of the cysteine-rich repeats of LDL receptor at the expected positions in LSGLR. The results presented are consistent with the hypothesis that all seven repeats in the ligand binding domain of LDL receptor may have been lifted directly from an ancestral gene instead of being evolutionary duplications of a single repeat recruited by the primitive LDL receptor from another gene.