Expression of chromosomal proteins HMG-14 and HMG-17 in transformed human cells.

The relation between cellular phenotype and expression of chromosomal high mobility group proteins 14 and 17 (HMG-14 and HMG-17) has been examined in human cell lineages. Quantitation of HMG-14 and HMG-17 mRNA in several human cell lines revealed differences in both the steady state mRNA level and in the ratio of HMG-14 to HMG-17 mRNA. Analysis of phenotypically distinct derivatives of human bronchial epithelial cells revealed small differences between both the steady state mRNA levels and the relative amount of these proteins among the clonal variants. The effect of myeloid differentiation on the mRNA level of HMG-14 and HMG-17 was examined in the human promyelocytic leukemia cell line HL-60 following treatment with several granulocytic and monocytic differentiating agents. The ratio of HMG-17 mRNA to either HMG-14 or histone H4 mRNA varied among the cell phenotypes suggesting that phenotype switching may result in detectable alterations in the expression of the HMG-14 and HMG-17 genes. The data suggest that, although the ratio of HMG-14 to HMG-17 mRNA varies among human cell lines, these variations are relatively small.

Transcriptional regulation of insulin-like growth factor-binding protein-5 by prostaglandin E2 in osteoblast cells.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is an autocrine and paracrine factor that modulates the effects of IGFs. We examined the mechanisms that regulate IGFBP-5 synthesis by PGE2 in osteoblast-enriched cells from fetal rat calvaria (Ob cells). PGE2 at 1 microM for 2-8 h increased IGFBP-5 heterogeneous nuclear RNA levels and did not change the half-life of IGFBP-5 messenger RNA in Ob cells, suggesting that PGE2 stimulates IGFBP-5 transcription. To analyze the elements responsible for this effect, regions of the mouse IGFBP-5 promoter from -2695 to +120 bp were ligated into pGL-2-basic and transiently transfected into Ob cells. PGE2 caused a time- and dose-dependent increase in IGFBP-5 promoter activity. Further analysis revealed two potential PGE2-responsive regions in the -2695 to -1470 and the -989 to -332 fragments. The effect of PGE2 on IGFBP-5 messenger RNA and heterogeneous nuclear RNA levels was mimicked by forskolin and inhibited by the PKA inhibitor H-89, suggesting that part of the PGE2 effect was mediated through a cAMP-dependent pathway. H-89 also blocked basal and PGE2-stimulated IGFBP-5 promoter activities. We conclude that PGE2 regulates IGFBP-5 synthesis in Ob cells by transcriptional mechanisms. PKA-dependent pathways account for part of the effect of PGE2 on IGFBP-5 expression. Deletion analysis of the IGFBP-5 promoter suggests the presence of two PGE2-responsive regions.

Regulation of insulin-like growth factor I transcription by prostaglandin E2 in osteoblast cells.

Insulin-like growth factor I (IGF-I) is a widely expressed abundant autocrine and paracrine factor that regulates the proliferation and differentiation of a variety of cell types. Prostaglandin E2 (PGE2) is a potent stimulator of IGF-I synthesis in bone. We examined the regulation of IGF-I synthesis by PGE2 in osteoblast-enriched (Ob) cells from fetal rat calvaria. PGE2 treatment of Ob cells at 1 microM for 2 h resulted in a 5-fold increase in heterogeneous nuclear RNA levels, as measured by a reverse transcriptase-polymerase chain reaction assay, suggesting an increase in IGF-I gene transcription. RNase protection analysis was used to map the transcriptional start sites in the IGF-I gene that are used in Ob cells. Consistent with other extrahepatic tissues, initiation of transcription occurs primarily at three sites within the 5'-regions of exon 1 of the IGF-I gene. PGE2 treatment did not alter start site usage. The regions upstream of these transcriptional start sites were analyzed by transiently transfecting Ob cells with putative rat IGF-I promoter sequences ligated to a luciferase reporter gene. Constructs containing 1.4 kilobases of the 5'-regions regions of exons 1 and 2 had significant promoter activity. PGE2 treatment of transfected Ob cells increased luciferase activity 5-fold when a 1.4-kilobase exon 1 promoter fragment was tested. This increase in luciferase activity was time and dose dependent. Smaller regions of the exon 1 promoter sequence gave higher basal activity and were less responsive to PGE2. We conclude that regions involved in IGF-I regulation by PGE2 are contained within the IGF-I promoter.

Characterization of transgenic mice with an increased content of chromosomal protein HMG-14 in their chromatin.

Chromosomal protein HMG-14 is a ubiquitous nuclear protein that may modulate the chromatin structure of transcriptionally active genes. To gain insights into the cellular function of the HMG-14 protein, we generated two transgenic mouse lines carrying either two or six copies of the human HMG-14 gene. The transgenic mice express human HMG-14 mRNA and protein in all tissues examined at a level reflecting the increased gene dosage, suggesting that the HMG14 transgene contains all the control regions necessary for regulated gene expression. Expression of the human HMG-14 protein does not alter the expression of the endogenous mouse HMG-14 protein or its close homolog, protein HMG-17. The intracellular distribution of the exogenous human protein is indistinguishable from that of the endogenous mouse protein, resulting in a three-fold increase in the level of the chromatin-bound HMG-14. The transgenic mice had a higher incidence of epithelial cysts in their thymus than did control animals. We conclude that the cellular levels of HMG-14/-17 are determined by gene copy number, that the DNA fragment containing the gene and about 1,000 bp flanking its 5' and 3' ends contain most of the elements necessary for gene expression, that the upper limits of HMG-14 in chromatin are not stringently regulated, and that a three-fold increase in chromatin-bound protein cause only mild phenotypic changes in the transgenic mice.

Inhibition by corticosteroids of epidermal growth factor-induced recovery of cyclooxygenase after aspirin inactivation.

Cultures of vascular smooth muscle cells superfused with [14C]arachidonic acid synthesized the antiplatelet substance prostacyclin as the major cyclooxygenase product. Prostacyclin synthesis was inactivated by aspirin, which irreversibly acetylates cyclooxygenase. Aspirin-treated cells recovered within 2 h by a process that was blocked by cycloheximide but not by actinomycin D, and that required a serum component identified as epidermal growth factor (EGF). EGF-induced recovery of cyclooxygenase was greatly potentiated by type beta transforming growth factor (TGF-beta). Incubation with EGF and TGF-beta in the 0.1-1.0 nanomolar range stimulated cyclooxygenase recovery up to 20-fold without increasing [35S]methionine incorporation into other cell proteins. Induction of cyclooxygenase by EGF and TGF-beta also was prevented by cycloheximide but not by actinomycin D. EGF-dependent recovery was blocked by preincubation with dexamethasone (2 microM), an effect that was duplicated by pure lipocortin (2-4 micrograms/ml). Incubation of membrane preparations from these cells with EGF selectively activated phosphorylation of a 35-kDa cellular protein that comigrated with lipocortin. The results suggest that cyclooxygenase recovery in aspirin-inactivated vascular smooth muscle cells is mediated by an EGF-dependent translational control that is inhibited by corticosteroids. The findings also provide a new mechanism whereby corticosteroids suppress inflammatory prostaglandins.

Aberrant expression of high mobility group chromosomal protein 14 affects cellular differentiation.

High mobility group (HMG) 14 is a ubiquitous chromosomal protein that binds specifically to nucleosomal DNA and may be involved in a process that confers distinct properties to the chromatin structure of transcriptionally active genes. To explore the involvement of this protein in regulation of gene expression, we studied the effect of aberrant expression of HMG-14 protein on cellular differentiation. We produced stably transfected C2C12 mouse myoblasts expressing the human HMG-14 protein under the control of the mouse mammary tumor virus promoter. Transformed colonies retained their potential do differentiate into myotubes. Induction of human HMG-14 expression by dexamethasone inhibited the myogenic process. Revertant colonies, which lost the ability to express human HMG-14, regained the ability to differentiate into myotubes. Inhibition of myoblast differentiation by aberrantly expressed HMG-14 correlated with down-regulation of myogenic determination factors. The results suggest that proper cellular differentiation requires regulated expression of HMG-14 protein and are consistent with the possibility that this protein may be involved in gene regulation.

Expression and synthesis of high mobility group chromosomal proteins in different rat skeletal cell lines during myogenesis.

The synthesis, turnover, and expression of all the major high mobility group (HMG) chromosomal proteins was studied in different rat skeletal myogenic cell lines. Whereas pulse-chase experiments revealed a similar half-life (greater than 2 cell generations) for all the HMG proteins in both L8 myoblasts and myotubes, [3H]lysine incorporation data indicated a 2- to 4-fold greater incorporation of the label in the HMG proteins in proliferating myoblasts relative to the nondividing myotubes. Analysis of the HMG-1, -14, and -17 mRNAs during myogenesis showed a significant down-regulation in L6 and L8 myotubes compared to the myoblasts. However, the timing of the shift and the extent of down-regulation was cell type-dependent, being more pronounced in L6 myotubes at fusion compared to 4 days postfusion in L8 myotubes. By contrast, L8-derived fusion-defective fu-1 cells over the same period of growth showed no change in HMG-14/17 mRNA levels. HMG-I(Y) protein isoforms, noted for the first time in rat myoblasts, like their counterparts, seemed to be stable and showed a precipitous reduction in their mRNAs during myogenesis. The results suggest a cell type-specific correlation between HMG expression and cell proliferation; they also argue for their role in maintenance of the cell's state of differentiation.

Cellular and clinical perspectives on skeletal insulin-like growth factor I.

Insulin-like growth factor (IGF) I, a polypeptide synthesized by skeletal cells, is presumed to act as an autocrine regulator of bone formation. IGF I stimulates bone replication of preosteoblastic cells and enhances the differentiated function of the osteoblast. The synthesis of skeletal IGF I is regulated by systemic hormones, most notably parathyroid hormone and glucocorticoids, as well as by locally produced factors, such as prostaglandins and other skeletal growth factors. Whereas hormones and growth factors regulate IGF I synthesis, the exact level of regulation has not been established and may involve both transcriptional and posttranscriptional mechanisms. The IGF I gene contains six exons, and both exon 1 and 2 contain transcription initiation sites. Extrahepatic tissues, including bone, express exon 1 transcripts, and regulation of the exon 1 promoter activity in osteoblasts is currently under study. It is apparent that the regulation of IGF I gene transcription as well as the regulation of mRNA stability is complex and tissue specific. It is possible that abnormalities in skeletal IGF I synthesis or activity play a role in the pathogenesis of bone disorders. In view of its important anabolic actions in bone, it is tempting to postulate the use of IGF I for the treatment of disorders characterized by decreased bone mass. An alternative could be the stimulation of the local production of IGF I in bone.

Immunochemical analysis of the exposure of high mobility group protein 14 and 17 surfaces in chromatin.

Antisera were elicited against synthetic peptides corresponding either to regions common to all members of the high mobility group 14 and 17 protein family protein or to distinct domains of the HMG-14 or HMG-17 subgroup. The antisera were used to probe the accessibility of various HMG domains in chromatin. Competitive enzyme-linked immunosorbent assays indicate that the central region of the proteins, which contains their DNA binding domain and is positively charged, is exposed to a smaller degree than the C-terminal region of the proteins, which has a net negative charge. The C-terminal regions of the HMG-14 and HMG-17 proteins are exposed and available to interact with other proteins.

Persistence of chromosomal proteins HMG-14/-17 in myotubes following differentiation-dependent reduction of HMG mRNA.

The expression of chromosomal proteins HMG-14 and HMG-17 during cellular differentiation was studied in cultured mouse myoblasts. During myogenesis the level of both HMG-14 and HMG-17 mRNA decreased to less than 20% of that found in myoblasts. The down-regulation of HMG-14/-17 mRNA occurred simultaneously with activation of muscle-specific actin mRNA and was not linked to DNA synthesis, indicating that it is a differentiation-, rather than a cell cycle-related event. Incorporation of radiolabeled lysine into HMG proteins was similar to that into the major histone fractions in that it was significant in myoblasts and undetectable in myotubes. The decrease in mRNA and protein synthesis did not affect the cellular levels of HMG protein. These results indicate that the regulation of HMG-14/-17 mRNA levels is different from that of the histones and is linked to differentiation rather than to DNA synthesis.

Cortisol inhibits the synthesis of insulin-like growth factor-binding protein-5 in bone cell cultures by transcriptional mechanisms.

Glucocorticoids inhibit the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5) in osteoblasts, but the mechanisms involved are unknown. IGFBP-5 stimulates bone cell growth, and its inhibition by glucocorticoids may be relevant to the action of this binding protein on bone formation. We tested the effects of cortisol on IGFBP-5 expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). Cortisol decreased IGFBP-5 polypeptide levels in the extracellular matrix and caused a time- and dose-dependent decrease in IGFBP-5 mRNA. IGFBP-5 transcripts were markedly decreased by cycloheximide, and further suppressive effects of cortisol could not be determined. Cortisol did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells. Cortisol decreased IGFBP-5 hnRNA, the rate of IGFBP-5 transcription, and the activity of the murine IGFBP-5 promoter by 35% in transient transfection experiments. Deletion analysis showed that the region responsive to cortisol is from base pairs -70 to +22, and E-box-binding proteins or c-Myb-related nuclear factors may be involved in its regulation. In conclusion, cortisol inhibits IGFBP-5 transcription in Ob cells through the Myb-binding domain. This effect may be partly responsible for the effect of glucocorticoids on bone formation.

Recombinant human chromosomal proteins HMG-14 and HMG-17.

Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.