Downregulation of repressive CUP/AP-2 isoforms during adipocyte differentiation.

Activation of transcription of the C/EBPalpha (CCAAT/enhancer binding protein alpha) gene is a critical event in the differentiation of 3T3-L1 preadipocytes into adipocytes. The kinetics of this process parallels a decline of AP-2alpha protein (also referred to as CUP, C/EBP undifferentiated protein) and decreased binding of CUP/AP-2alpha to the C/EBPalpha promoter. Mutation of the CUP/AP-2 binding sites in the C/EBPalpha promoter results in increased C/EBPalpha expression. Based on these findings, it appears that decline in AP-2alpha expression is an important early event in the adipocyte differentiation program. In the studies presented here, we identify three mRNAs that encode the repressive CUP/AP-2alpha isoforms expressed in undifferentiated 3T3-L1 preadipocytes. We demonstrate that the kinetics of the decline of these isoforms' expression over the course of differentiation parallels both the decrease in CUP/AP-2alpha DNA binding activity and the increase in C/EBPalpha protein observed in previous studies.

Parathyroid hormone-related peptide is produced by cultured cerebellar granule cells in response to L-type voltage-sensitive Ca2+ channel flux via a Ca2+/calmodulin-dependent kinase pathway.

Parathyroid hormone (PTH)-related peptide (PTHrP) is expressed in the adult mammalian brain, but its function is unknown. Here we show that PTHrP and the PTH/PTHrP receptor are products of cerebellar granule cells in primary culture. Granule cells maintained under depolarizing conditions (25 mM K+) make and release PTHrP. Further, PTHrP-(1-36) stimulates cAMP accumulation in granule neurons in a dose-dependent manner with half-maximal activation at approximately 16 nM. Granule cell PTHrP mRNA is activity-dependent, and the pathway of regulation depends absolutely on the flux of Ca2+ ions through the L-type voltage-sensitive Ca2+ channel and the Ca2+/calmodulin kinase cascade. PTHrP is therefore a neuropeptide whose regulation depends upon L-type voltage-sensitive Ca2+ channel activity, and the gene is expressed under conditions that promote granule cell survival.

Regulation of parathyroid hormone-related peptide gene expression by estrogen in GH4C1 rat pituitary cells has the pattern of a primary response gene.

The parathyroid hormone-related peptide (PTHrP) gene has been reported to be subject to a wide variety of physiological and pharmacological controls. Two distinct patterns of PTHrP mRNA response have been recognized, one characterized by a prolonged or plateau response lasting many hours to days and the second characterized by rapid induction-deinduction kinetics and lasting 1 to several hours. The kinetics of the second pattern are similar to those displayed by primary response genes like nuclear protooncogenes, cytokines, and growth factors. In GH4C1 rat pituitary cells, 17 beta-estradiol induced a rapid and transient increase in PTHrP mRNA expression, with a peak response at 1-2 h. This response appeared to be due to a rapid and transient burst in gene transcription, which by runoff analysis was maximal at 20-40 min and declined thereafter. PTHrP mRNA half-life was 30 min in these cells and was unaltered by estradiol. Cycloheximide did not block the 17 beta-estradiol-induced response but rather prolonged it, and runoff analysis revealed that this effect was due to a prolongation or persistence of PTHrP gene transcription. These findings suggest that the transient nature of the native response reflects the effects of an estrogen-inducible repressor. All of these features are characteristic of a prototypical primary response gene.

Region-specific methylation of the parathyroid hormone-related peptide gene determines its expression in human renal carcinoma cell lines.

Tumor production of a parathyroid hormone-related peptide (PTHrP) is a common cause of the syndrome of humoral hypercalcemia of malignancy, which is frequently associated with renal cell carcinomas. Why certain renal cell carcinomas produce PTHrP while others do not is unknown. Using a system of 12 human renal carcinoma cell lines which either do (n = 6) or do not (n = 6) produce PTHrP, we found that the expression of the PTHrP gene in these cell lines is controlled at the transcriptional level. Transfection studies failed to demonstrate variation in PTHrP promoter activity in these cell lines sufficient to account for the differential PTHrP expression, implicating a cis-acting mechanism. Transcription of the PTHrP gene in these cell lines was found to correlate with the methylation state of specific CpG dinucleotides located within the promoter region but outside a CpG island. The functional importance of this mechanism of control was confirmed by the ability of the demethylating agent, 5-azacytidine, to induce PTHrP mRNA expression in previously nonexpressing cell lines.

Erythrocyte invasion by two Plasmodium falciparum isolates differing in sialic acid dependency in the presence of glycophorin A antibodies.

Merozoites of Plasmodium falciparum depend on glycophorins for invasion into human erythrocytes, although this dependency varies between different geographic isolates of the species. The FCR-3 (Gambia) isolate appears to be fully dependent on the N-acetylneuraminic acid (NeuNAc) residues of the O-linked tetrasaccharide of glycophorin for invasion. Invasion of the CDC-1 (Honduras) isolate into neuraminidase treated erythrocytes is 50% of that into normal erythrocytes. This and additional results suggest that this isolate is not fully dependent on the O-linked saccharides of glycophorin. In the present study, invasion of CDC-1 and FCR-3 isolates into erythrocytes was examined in the presence of Fab fragments of monoclonal antibodies directed against different domains of glycophorin. Fab fragments directed against the carbohydrate domain inhibited invasion of both isolates but inhibited invasion by the FCR-3 isolate more than CDC-1 isolate. The reactivity of monoclonal antibodies (Mabs) directed against the carbohydrate domain was dependent on the NeuNAc residues as binding was abolished in neuraminidase treated erythrocytes. Mabs directed against the peptide domain of glycophorin A did not significantly inhibit invasion by either isolate. These results are consistent with other findings that the CDC-1 isolate is not fully dependent on the carbohydrate domain of glycophorin for invasion.

Erythrocyte receptor recognition varies in Plasmodium falciparum isolates.

N-Acetylneuraminic acid (NeuNAc) is the terminal sugar residue of the O-linked tetrasaccharide linked to erythrocyte sialoglycoproteins, glycophorins. Erythrocytes lacking NeuNAc have been shown previously to be resistant to invasion by certain isolates of Plasmodium falciparum merozoites. We report here variation between different geographic isolates of P. falciparum in their dependency on NeuNAc for invasion of host erythrocytes. Seven different geographic isolates of P. falciparum were examined for their ability to invade neuraminidase treated erythrocytes. For all isolates invasion was reduced significantly, although considerable variation in NeuNAc dependency was apparent. Three isolates, FCR-3, FVO and It2, exhibited a very high dependence on NeuNAc residues for invasion (invasion reduced greater than 90%), whereas two isolates (Thai-Tn and FC-27) exhibited a moderately high dependence (invasion reduced 75%). Two other isolates (CDC-1 and 7G8) exhibited moderate dependence on NeuNAc (invasion reduced 50%). Cleavage of the complete O-linked tetrasaccharide by O-glycanase removes all carbohydrate from glycophorin A, B and C except the single N-linked oligosaccharide on glycophorin A and C. Invasion of FCR-3 and CDC-1 isolates into O-glycanase treated erythrocytes was not markedly different from that into neuraminidase treated cells indicating that NeuNAc is the important residue of the tetrasaccharide for both isolates. Invasion into endo-beta-galactosidase treated erythrocytes, in which the lactosaminoglycan side chain of band 3 and band 4.5 is cleaved, was not significantly reduced for either the CDC-1 or FCR-3 isolates. Additional results on the trypsin insensitivity of band 3 also suggest that this erythrocyte protein is not important in P. falciparum recognition. The greatest divergence in receptor specificity between FCR-3 and CDC-1 isolates was apparent in invasion into periodate-treated erythrocytes. Periodate oxidation results in cleavage of the exocyclic hydroxyl groups of the terminal NeuNAc but leaves its COOH group unaltered. These experiments also illustrated that the negatively charged COOH group of NeuNAc is not the important group in the interaction of the merozoite with the NeuNAc. Trypsin-treated erythrocytes were almost fully resistant to invasion by CDC-1 as well as the FCR-3 isolates suggesting that the CDC-1 isolate, in addition to interacting with NeuNAc, depends on a trypsin sensitive site for invasion. This site could involve the N-linked saccharide on glycophorin A and C or a protein on the erythrocyte surface unrelated to the glycophorins.