Increased metabolic rate and insulin sensitivity in male mice lacking the carcino-embryonic antigen-related cell adhesion molecule 2.

AIMS/HYPOTHESIS: The carcino-embryonic antigen-related cell adhesion molecule (CEACAM)2 is produced in many feeding control centres in the brain, but not in peripheral insulin-targeted tissues. Global Ceacam2 null mutation causes insulin resistance and obesity resulting from hyperphagia and hypometabolism in female Ceacam2 homozygous null mutant mice (Cc2 [also known as Ceacam2](-/-)) mice. Because male mice are not obese, the current study examined their metabolic phenotype. METHODS: The phenotype of male Cc2(-/-) mice was characterised by body fat composition, indirect calorimetry, hyperinsulinaemic-euglycaemic clamp analysis and direct recording of sympathetic nerve activity. RESULTS: Despite hyperphagia, total fat mass was reduced, owing to the hypermetabolic state in male Cc2(-/-) mice. In contrast to females, male mice also exhibited insulin sensitivity with elevated ß-oxidation in skeletal muscle, which is likely to offset the effects of increased food intake. Males and females had increased brown adipogenesis. However, only males had increased activation of sympathetic tone regulation of adipose tissue and increased spontaneous activity. The mechanisms underlying sexual dimorphism in energy balance with the loss of Ceacam2 remain unknown. CONCLUSIONS/INTERPRETATION: These studies identified a novel role for CEACAM2 in the regulation of metabolic rate and insulin sensitivity via effects on brown adipogenesis, sympathetic nervous outflow to brown adipose tissue, spontaneous activity and energy expenditure in skeletal muscle.

Decreased osteoclastogenesis and high bone mass in mice with impaired insulin clearance due to liver-specific inactivation to CEACAM1.

Type 2 diabetes is associated with normal-to-higher bone mineral density (BMD) and increased rate of fracture. Hyperinsulinemia and hyperglycemia may affect bone mass and quality in the diabetic skeleton. In order to dissect the effect of hyperinsulinemia from the hyperglycemic impact on bone homeostasis, we have analyzed L-SACC1 mice, a murine model of impaired insulin clearance in liver causing hyperinsulinemia and insulin resistance without fasting hyperglycemia. Adult L-SACC1 mice exhibit significantly higher trabecular and cortical bone mass, attenuated bone formation as measured by dynamic histomorphometry, and reduced number of osteoclasts. Serum levels of bone formation (BALP) and bone resorption markers (TRAP5b and CTX) are decreased by approximately 50%. The L-SACC1 mutation in the liver affects myeloid cell lineage allocation in the bone marrow: the (CD3(-)CD11b(-)CD45R(-)) population of osteoclast progenitors is decreased by 40% and the number of (CD3(-)CD11b(-)CD45R(+)) B-cell progenitors is increased by 60%. L-SACC1 osteoclasts express lower levels of c-fos and RANK and their differentiation is impaired. In vitro analysis corroborated a negative effect of insulin on osteoclast recruitment, maturation and the expression levels of c-fos and RANK transcripts. Although bone formation is decreased in L-SACC1 mice, the differentiation potential and expression of the osteoblast-specific gene markers in L-SACC1-derived mesenchymal stem cells (MSC) remain unchanged as compared to the WT. Interestingly, however, MSC from L-SACC1 mice exhibit increased PPARgamma2 and decreased IGF-1 transcript levels. These data suggest that high bone mass in L-SACC1 animals results, at least in part, from a negative regulatory effect of insulin on bone resorption and formation, which leads to decreased bone turnover. Because low bone turnover contributes to decreased bone quality and an increased incidence of fractures, studies on L-SACC1 mice may advance our understanding of altered bone homeostasis in type 2 diabetes.

Androgen regulation of the cell-cell adhesion molecule-1 (Ceacam1) gene.

Previous studies have established that the cell-cell adhesion molecule-1 (CEACAM1, previously known as C-CAM1) functions as a tumor suppressor in prostate cancer and is involved in the regulation of prostate growth and differentiation. However, the molecular mechanism that modulates CEACAM1 expression in the prostate is not well defined. Since the growth of prostate epithelial cells is androgen-regulated, we investigated the effects of androgen and the androgen receptor (AR) on CEACAM1 expression. Transient transfection experiments showed that the AR can enhance the Ceacam1 promoter activity in a ligand-dependent manner and that the regulatory element resides within a relatively short (-249 to -194 bp) segment of the 5'-flanking region of the Ceacam1 gene. This androgen regulation is likely through direct AR-promoter binding because a mutant AR defective in DNA binding failed to upregulate reporter gene expression. Furthermore, electrophoretic mobility shift assays demonstrated that the AR specifically binds to this sequence, and mutation analysis of the potential ARE sequences revealed a region within the sequence that was required for the AR to activate the Ceacam1 gene. Therefore, the regulation of Ceacam1 gene expression by androgen may be one of the mechanisms by which androgen regulates prostatic function.

Differences in tissue-specific and embryonic expression of mouse Ceacam1 and Ceacam2 genes.

The intercellular adhesion molecule CEACAM1, also known as C-CAM1 (where CAM is cell-adhesion molecule), can function as a tumour suppressor in several carcinomas, including those of the prostate, breast, bladder and colon. This suggests that CEACAM1 may play an important role in the regulation of normal cell growth and differentiation. However, there is no direct evidence to support this putative function of CEACAM1. To elucidate its physiological function by targeted gene deletion, we isolated the Ceacam genes from a mouse 129 Sv/Ev library. Although there is only one Ceacam1 gene in humans and one in rats, two homologous genes (Ceacam1 and Ceacam2) have been identified in the mouse. Our sequence analysis revealed that the genes encoded nine exons and spanned approx. 16-17 kb (Ceacam1) and 25 kb (Ceacam2). The genes were highly similar (79.6%). The major differences in the protein-coding regions were located in exons 2, 5 and 6 (76.9%, 87.0% and 78.5% similarity respectively). In addition, introns 2, 5 and 7 were also significantly different, being 29.7%, 59.8% and 64.5% similar respectively. While most of these differences were due to nucleotide substitutions, two insertions of 418 and 5849 bp occurred in intron 2 of Ceacam2, and another two insertions of 1384 and 197 bp occurred in introns 5 and 7 respectively. To determine whether functional redundancy exists between Ceacam1 and Ceacam2, we examined their expression in 16 mouse tissues by using semi-quantitative reverse transcription-PCR. As in human and rat, in the mouse Ceacam1 mRNA was highly abundant in the liver, small intestine, prostate and spleen. In contrast, Ceacam2 mRNA was only detected in kidney, testis and, to a lesser extent, spleen. Reverse transcription-PCR using testis RNA indicated that Ceacam2 in the testis is an alternatively spliced form containing only exons 1, 2, 5, 6, 8 and 9. In the mouse embryo, Ceacam1 mRNA was detected at day 8.5, disappeared between days 9.5 and 12.5, and re-appeared at day 19. On the other hand, no Ceacam2 mRNA was detected throughout embryonic development. The different tissue expression patterns and regulation during embryonic development suggest that the CEACAM1 and CEACAM2 proteins, although highly similar, may have different functions both during mouse development and in adulthood.

Sucrase-alpha-dextrinase in the spontaneously diabetic BioBreed Wistar rat: altered intracellular carbohydrate processing.

Sucrase-alpha-dextrinase (S-D), a glycoprotein hydrolase in the border surface of the enterocyte, is altered in congenitally diabetic BioBreed Wistar (BB(d)) rats. Its intracellular assembly was examined in the current studies. Following pulse-chase experiments, S--D was specifically immuno-isolated from ER-Golgi and brush border membranes, and examined by SDS-PAGE and autoradiography. While synthesis and co-translational glycosylation of an immature species, P(i), in the ER proceeded normally, post-translational maturation of the N-linked carbohydrate chains was altered in the BB(d) rat. The mature species, P(m), was 10 kDa larger in BB(d) than in normal rats, and approximately 25% of its N-linked chains remained immature. The difference in mass was attributed to an appreciably larger mass of the O-linked chains of P(m) in BB(d) rats. In vivo kinetics of intracellular assembly displayed a delay in the appearance of P(m) in Golgi (Wistar, 15 min; BB(d), 30--60 min). These experiments, revealing structural alterations in congenital diabetes suggest an important role for intracellular glycosylation in the orderly assembly and processing of brush border S-D in the enterocyte.

Intestinal aminooligopeptidase in diabetic BioBreed rat: altered posttranslational processing and trafficking.

The structure of aminooligopeptidase (AOP), an intestinal brush-border digestive hydrolase, is abnormal in human diabetes and in the congenitally diabetic BioBreed Wistar (BB(d)) rat. Its assembly in the BB(d) rat was examined. After normal initial synthesis and assembly of immature AOP precursor (AOP(i)) with high-mannose N-linked chains in the endoplasmic reticulum (ER), processing of N-linked glycans in Golgi yielded a smaller than normal mature AOP precursor (AOP(m)) with persistence of some high-mannose N-linked chains. Deglycosylation analyses suggested that the mass difference could be attributed to a lower mass of N-linked with unaltered O-linked glycans in AOP(m) of the diabetic rat. Intrajejunal pulse-chase experiments revealed that the conversion of AOP(i) to AOP(m) occurred at 30 min of chase in normal rats but at 60-90 min in diabetic rats, reflecting delay in ER-to-Golgi transport or a slower processing of high-mannose chains. Once maximal transport to Golgi was achieved, the residence time in Golgi was shortened in diabetes. This altered processing of the precursor accounted for the altered structure of AOP in diabetes.

The differential effects of pp120 (Ceacam 1) on the mitogenic action of insulin and insulin-like growth factor 1 are regulated by the nonconserved tyrosine 1316 in the insulin receptor.

pp120 (Ceacam 1) undergoes ligand-stimulated phosphorylation by the insulin receptor, but not by the insulin-like growth factor 1 receptor (IGF-1R). This differential phosphorylation is regulated by the C terminus of the beta-subunit of the insulin receptor, the least conserved domain of the two receptors. In the present studies, deletion and site-directed mutagenesis in stably transfected hepatocytes derived from insulin receptor knockout mice (IR(-/-)) revealed that Tyr(1316), which is replaced by the nonphosphorylatable phenylalanine in IGF-1R, regulated the differential phosphorylation of pp120 by the insulin receptor. Similarly, the nonconserved Tyr(1316) residue also regulated the differential effect of pp120 on IGF-1 and insulin mitogenesis, with pp120 downregulating the growth-promoting action of insulin, but not that of IGF-1. Thus, it appears that pp120 phosphorylation by the insulin receptor is required and sufficient to mediate its downregulatory effect on the mitogenic action of insulin. Furthermore, the current studies revealed that the C terminus of the beta-subunit of the insulin receptor contains elements that suppress the mitogenic action of insulin. Because IR(-/-) hepatocytes are derived from liver, an insulin-targeted tissue, our observations have finally resolved the controversy about the role of the least-conserved domain of insulin and IGF-1Rs in mediating the difference in the mitogenic action of their ligands, with IGF-1 being more mitogenic than insulin.

Metabolic regulation of Na(+)/P(i)-cotransporter-1 gene expression in H4IIE cells.

We showed that the rat Na(+)/P(i) cotransporter-1 (RNaPi-1) gene was regulated by insulin and glucose in rat hepatocytes. The aim of this work was to elucidate signaling pathways of insulin-mediated metabolic regulation of the RNaPi-1 gene in H4IIE cells. Insulin increased RNaPi-1 mRNA abundance in the presence of glucose and decreased RNaPi-1 mRNA in the absence of glucose, clearly establishing an involvement of metabolic signals for insulin-induced upregulation of the RNaPi-1 gene. Pyruvate and insulin increased RNaPi-1 expression but downregulated L-pyruvate kinase, indicating the existence of gene-specific metabolic signals. Although fructose, glycerol, and lactate could support insulin-induced upregulation of the RNaPi-1 gene, compounds entering metabolism beyond pyruvate oxidation, such as acetate and citrate, could not, suggesting that RNaPi-1-specific metabolic signals are generated at or above pyruvate oxidation. Wortmannin, LY-294002, and rapamycin abolished the insulin effect on the RNaPi-1 gene, whereas expression of dominant negative Asn(17) Ras and mitogen-activating protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 exhibited no effect. Thus we herein propose that metabolic regulation of RNaPi-1 expression by insulin is mediated through the phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase pathways, but not the Ras/MAPK pathway.

Frequency of tracheobronchial foreign bodies in children and adolescents.

OBJECTIVE: The problem of foreign body aspiration in the community has been studied and compared with other reports. METHODS: We have retrospectively studied patients who had bronchoscopy for suspected foreign bodies in the tracheobronchial tree, attending or referred to Al-Noor Specialist Hospital, Makkah over a period of 3 years from May 1996 to May 1999. RESULTS: The total number of patients was 94 (62 male and 32 female). Ages ranged from 4 months to 45 years(mean age 3.77 years), 85% of children being under the age of 5 years. One hundred bronchoscopies (6 repeat bronchoscopies) and one thoracotomy were carried out. Foreign bodies were removed from 60 patients (64%). Six (10%) did not have any definite history, while 15 patients (21%) with definite history of foreign body aspiration had negative bronchoscopy. An aspirated Fis-Fis (Alfalfa, Lucerne) seed accounted for more than one-third of all foreign bodies. The most frequent symptoms, signs, radiological findings and site of foreign body lodgement in the tracheobronchial tree are discussed. CONCLUSION: We conclude that a negative history, clinical examination and chest x-ray do not necessarily exclude aspirated foreign body material. Bronchoscopy is the most effective diagnostic and therapeutic modality to prevent complications related to neglected foreign body aspiration. In addition to children, teenagers and adolescents are also not immune to this problem. We recommend early referral to an appropriate hospital on suspicion or if symptoms persist. However, preventive measures, remain the best means of protecting these children.

Comparison of the intracellular trafficking of two alternatively spliced isoforms of pp120, a substrate of the insulin receptor tyrosine kinase.

pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein in the hepatocyte. It is expressed as two spliced isoforms differing by the presence (full length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Because the two isoforms differ by their ability to regulate receptor-mediated insulin endocytosis and degradation, we aimed to investigate the cellular basis for this functional difference by comparing their intracellular trafficking. During its intracellular assembly, pp120 is transported from the trans-Golgi network to the sinusoidal domain of the plasma membrane before its final transcytosis to the bile canalicular domain. Because both isoforms are expressed in hepatocytes, we examined their intracellular trafficking in NIH 3T3 fibroblasts individually transfected with each isoform. Pulse-chase experiments demonstrated that most of the newly synthesized full-length isoform reached complete maturation at about 60 min of chase. By contrast, only about 40% of the newly synthesized truncated isoform underwent complete maturation, even at more prolonged chase. Moreover, a significant portion of the truncated isoform appeared to be targeted to lysosomes. Abolishing basal phosphorylation on Ser(503) by cAMP-dependent serine kinase by mutating this residue to alanine was correlated with incomplete maturation of full length pp120 in NIH 3T3 cells and hepatocytes. This finding suggests that the intracellular domain of pp120 contains information that regulates its vectorial sorting from the trans-Golgi network to the plasma membrane.

Cell adhesion properties and effects on receptor-mediated insulin endocytosis are independent properties of pp120, a substrate of the insulin receptor tyrosine kinase.

pp120 undergoes phosphorylation by the tyrosine kinase of the insulin, not the insulin-like growth factor 1 (IGF-1), receptor. Moreover, pp120 stimulates receptor-mediated insulin, but not IGF-1, endocytosis, suggesting that pp120 phosphorylation underlies its effect on insulin endocytosis. pp120 phosphorylation also underlies its bile acid transport and tumor suppression functions. In addition to depending on the intracellular tail, the cell adhesion property of pp120 depends on Arg98 in the N-terminal IgV-like ectoplasmic domain. To investigate whether this domain mediates the effect of pp120 on insulin endocytosis, we mutated Arg98 to Ala and examined whether this mutation altered pp120 phosphorylation and its effect on ligand endocytosis in transfected NIH 3T3 cells. This mutation did not modify either pp120 phosphorylation or its effect on receptor-mediated ligand endocytosis. These findings support the hypothesis that stimulation of insulin endocytosis by pp120 is not mediated by Arg98 in the N-terminal IgV-like ectoplasmic domain of pp120.

Persistent expression of foreign genes in cultured hepatocytes: expression vectors.

We have optimized a liposome-based transfection method that mediated highly efficient stable expression of foreign genes in hepatocytes. Moreover, we have observed that the metallothionein 1 promoter in the bovine papilloma virus-based expression vector drove the highest expression of foreign genes in hepatocytes as compared with the cytomegalovirus and the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoters in the pcDNA 3-based expression vector. The cytomegalovirus promoter failed to yield significant expression in these cells. Furthermore, expression of foreign genes persisted up to at least 15 passages when expression was under the control of either the EF-1alpha or the metallothionein 1 promoter. Thus, these two promoters led to comparable stability of foreign genes in hepatocytes, with the metallothionein 1 promoter yielding a higher level of expression of foreign genes in these cells.

Insulin stimulates pp120 endocytosis in cells co-expressing insulin receptors.

pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein that is expressed in the hepatocyte as two spliced isoforms differing by the presence (full-length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Co-expression of full-length pp120, but not its phosphorylation-defective isoforms, increased receptor-mediated insulin endocytosis and degradation in NIH 3T3 fibroblasts. We, herein, examined whether internalization of pp120 is required to mediate its effect on insulin endocytosis. The amount of full-length pp120 expressed at the cell surface membrane, as measured by biotin labeling, markedly decreased in response to insulin only when insulin receptors were co-expressed. In contrast, when phosphorylation-defective pp120 mutants were co-expressed, the amount of pp120 expressed at the cell surface did not decrease in response to insulin. Indirect immunofluorescence analysis revealed that upon insulin treatment of cells co-expressing insulin receptors, full-length, but not truncated, pp120 co-localized with alpha-adaptin in the adaptor protein complex that anchors endocytosed proteins to clathrin-coated pits. This suggests that full-length pp120 is part of a complex of proteins required for receptor-mediated insulin endocytosis and that formation of this complex is regulated by insulin-induced pp120 phosphorylation by the receptor tyrosine kinase. In vitro GST binding assays and co-immunoprecipitation experiments in intact cells further revealed that pp120 did not bind directly to the insulin receptor and that its association with the receptor may be mediated by other cellular proteins.

pp120, a substrate of the insulin receptor tyrosine kinase, is associated with phosphatase activity.

pp120, a plasma membrane glycoprotein, is phosphorylated on Tyr488 by the insulin receptor tyrosine kinase. This requires basal phosphorylation on Ser503 by cAMP-dependent serine kinase. Phe513, the other tyrosine residue in the intracellular domain, does not undergo insulin-stimulated phosphorylation. Phe488 mutation abolished basal and insulin-stimulated phosphorylation, whereas, Phe513 plus Phe488 mutation markedly decreased the effect of insulin on pp120 phosphorylation without altering basal phosphorylation in intact cells. To investigate whether basal phosphorylation of pp120 is regulated by a phosphatase activity that requires Tyr513, in vitro phosphorylation assays using partially purified glycoproteins from stably transfected NIH 3T3 cells were performed in the absence of phosphatase inhibitors. Wild-type pp120 was promptly dephosphorylated, whereas, Y513F pp120 was not. Decreasing pp120 expression by antisense cDNA transfection proportionally decreased phosphatase activity in H4-II-E hepatoma cells measured by the p-nitrophenyl phosphate assay. This suggests that pp120 is associated with phosphatase activity that requires an intact Tyr513 residue.

Effect of pp120 on receptor-mediated insulin endocytosis is regulated by the juxtamembrane domain of the insulin receptor.

pp120, a substrate of the insulin receptor tyrosine kinase, does not undergo ligand-stimulated phosphorylation by the insulin-like growth factor-1 (IGF-1) receptor. However, replacement of the C-terminal domain of the IGF-1 receptor beta-subunit with the corresponding segment of the insulin receptor restored pp120 phosphorylation by the chimeric receptor. Since pp120 stimulates receptor-mediated insulin endocytosis when it is phosphorylated, we examined whether pp120 regulates IGF-1 receptor endocytosis in transfected NIH 3T3 cells. pp120 failed to alter IGF-1 receptor endocytosis via either wild-type or chimeric IGF-1 receptors. Thus, the effect of pp120 on hormone endocytosis is specific to insulin, and the C-terminal domain of the beta-subunit of the insulin receptor does not regulate the effect of pp120 on insulin endocytosis. Mutation of Tyr960 in the juxtamembrane domain of the insulin receptor abolished the effect of pp120 to stimulate receptor endocytosis, without affecting pp120 phosphorylation by the insulin receptor. These findings suggest that pp120 interacts with two separate domains of the insulin receptor as follows: a C-terminal domain required for pp120 phosphorylation and a juxtamembrane domain required for internalization. We propose that the interaction of pp120 with the juxtamembrane domain is indirect and requires one or more substrates that bind to Tyr960 in the insulin receptor.

Differential effect of pp120 on insulin endocytosis by two variant insulin receptor isoforms.

The insulin receptor is expressed as two variably spliced isoforms that differ by the absence (isoform A) or presence (isoform B) of a 12-amino acid sequence encoded by exon 11 at the carboxy terminus of the alpha-subunit. Coexpression of the A isoform and pp120, a substrate of the insulin receptor tyrosine kinase, in NIH 3T3 fibroblasts increased receptor A-mediated insulin endocytosis and degradation by two- to threefold compared with cells expressing receptors alone. Because B is the predominant isoform in the liver and binds insulin with lower affinity than A, we have examined the effect of pp120 on receptor B-mediated endocytosis. In contrast to isoform A, the effect of pp120 on isoform B-mediated insulin internalization and degradation in stably transfected NIH 3T3 cells was minimal.

Differential phosphorylation of pp120 by insulin and insulin-like growth factor-1 receptors: role for the C-terminal domain of the beta-subunit.

pp 120, a plasma membrane glycoprotein expressed by hepatocytes, is a substrate of the insulin receptor tyrosine kinase. Since insulin-like growth factor-1 (IGF-1) and insulin receptors are structurally homologous, we investigated whether pp120 is also a substrate of the IGF-1 receptor tyrosine kinase. IGF-1 receptor failed to phosphorylate pp120 in response to IGF-1 in stably transfected NIH 3T3 fibroblasts. However, replacement of the C-terminal domain of the beta-subunit of the IGF-1 receptor with the corresponding fragment in the insulin receptor restored ligand-stimulated pp120 phosphorylation, suggesting that this domain plays a regulatory role in pp120 phosphorylation. Since pp120 is the first identified substrate specific for the insulin vis-à-vis the IGF-1 receptor tyrosine kinase, the pp120 signaling pathway may constitute a novel mechanism for the distinct cellular effects of insulin and IGF-1, the former being principally involved in metabolism, and the latter in mitogenesis.

High density insulin receptor-positive T lymphocytes from nonobese diabetic mice transfer insulitis and diabetes.

In the nonobese diabetic mouse, insulin-dependent diabetes is an autoimmune disease characterized by T cell-mediated invasion and destruction of pancreatic islet beta cells. The importance of insulin receptor (IR) expression in the pathogenesis of diabetes was examined, since it has been shown that the IR is a chemotactic receptor capable of directing cell movement in response to insulin. Using polyclonal antisera to the IR, phenotypic analysis of purified splenic T cells from diabetic mice showed that about 15% of T cells expressed high density IR (IRhigh). In addition, IRhigh T cells were already a dominant phenotype in the insulitis of young prediabetic mice. To determine the ability of IRhigh T cells to transfer diabetes, cells were sorted by flow cytometry before adoptive transfer into young (6- to 8-wk-old) nondiabetic irradiated nonobese mice. Transfer of as few as 3 x 10(6) purified IRhigh T cells alone resulted in rapid onset of insulitis and diabetes, and IRhigh-depleted T cells were essentially unable to passage either insulitis or diabetes. The adoptive transfer of disease was not due to the transfer of activated cells, since removal of IL-2R+ or transferrin R+ cells did not alter diabetes transfer. Therefore, IRhigh T cells are aggressively diabetogenic, suggesting that increased IR expression may provide a mechanism for delivering potentially autoreactive T cells to the islet, regardless of their activation state.

Modifications of RNA processing modulate the expression of hemoglobin genes.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.