Steady-state Levels Of Alpha Research Articles

Retinoic acid stimulates expression of the functional osteoclast integrin alpha v beta 3: transcriptional activation of the beta 3 but not the alpha v gene.

The capacity of osteoclasts to resorb bone depends, in part, on the surface expression of the integrin alpha v beta 3. We have investigated whether the steroid hormone retinoic acid, known to stimulate bone resorption, regulates the appearance of the alpha v beta 3 complex in avian osteoclast presursors. Using surface labeling, followed by immunoprecipitation with a alpha v beta 3-specific antibody, we show that retinoic acid increases surface expression of the heterodimer in a dose- and time-dependent manner. Northern analysis reveals that the high basal steady-state levels of alpha v mRNA do not change, while those for beta 3 rise significantly from their initially low levels. Nuclear run-on studies confirm that steroid treatment stimulates transcription of the beta 3, but not the alpha v gene. Osteoclast precursors treated with retinoic acid exhibit increased multinucleation and expression of the osteoclast marker enzyme tartrate-resistant acid phosphatase. However, the fused cells do not have an increased capacity to resorb bone. In summary, multinucleated cells generated in this study do not represent fully differentiated bone-resorbing polykaryons. These results suggest that treatment of osteoclast precursors with retinoic acid is necessary, but insufficient, for expression of the mature osteoclast phenotype.

Transcriptional activation of the α1(1) procollagen gene in systemic sclerosis dermal fibroblasts. Role of intronic sequences

To investigate the transcriptional regulation of the alpha 1(I) procollagen gene (COL1A1) in cultured dermal fibroblasts from patients with diffuse systemic sclerosis (SSc) of recent onset and to evaluate the role that intronic sequences may play in the upregulated expression of COL1A1 in SSc dermal fibroblasts. Dermal fibroblasts from 6 patients with diffuse SSc of recent onset and from 3 healthy individuals were studied. The steady-state levels of alpha 1(I) procollagen messenger RNA were evaluated by Northern hybridization analysis, and the transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs containing portions of COL1A1 promoter (with 5' end points at -5.3 kb, -2.3 kb, and -804 bp and 3' end point at +42 bp) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. To examine the role of intronic sequences, constructs containing, in addition to the COL1A1 promoter, a portion of the first intron (+380 bp to +1,440 bp) cloned in front of the CAT gene were transfected. The efficiency of transfections was normalized relative to the net amount of CAT plasmid actually transfected into recipient cells, determined by a modified Southern hybridization procedure. Maximal CAT activity was observed with constructs extending from -804 bp to +42 bp in both normal and SSc fibroblasts. However, the activity driven by this construct was 80-110% higher in SSc fibroblasts. The CAT activity driven by a construct with a 5' end point at -5.3 kb was only 15-20% higher in SSc cells, and the CAT activity driven by a construct with a 5' end point -2.3 kb was 35-45% higher in SSc fibroblasts. The CAT activity driven by the -804-bp promoter construct was increased up to 4-fold in SSc fibroblasts in comparison with normal cells when the intronic segment spanning +380 bp to +1,440 bp was included in the transfected construct. The results directly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from SSc patients. The data also indicate that first-intron sequences of COL1A1 are required for maximal transcriptional activity of the collagen gene and may play an important role in the up-regulation of its expression in SSc fibroblasts.

Relative expression of the human epithelial Na+ channel subunits in normal and cystic fibrosis airways.

The availability of the newly cloned subunits (alpha, beta, gamma) of the epithelial Na+ channel (ENaC) permits molecular studies of the pathogenesis of the abnormal Na+ transport rates of cystic fibrosis (CF) airway epithelia. Northern analyses of airway epithelia showed that both normal and CF airway epithelia express ENaC subunit mRNAs in a ratio of alpha > beta > gamma. In situ hybridization studies revealed expression of all three ENaC subunits in the superficial epithelium and the alpha- and beta-subunits in the gland ductular and acinar epithelium of both normal and CF airways. Ribonuclease protection assays revealed that the steady-state levels of alpha-, beta-, and gamma-ENaC mRNAs were similar in CF and normal airway superficial epithelia. These findings indicate that 1) Na+ transport defects in CF airways disease may be expressed in glandular acinar and ductal epithelium as well as superficial epithelium, and 2) the molecular pathogenesis of Na+ hyperabsorption in CF airways does not reflect increased levels of Na+ channel mRNAs, and probably number, but reflects an absence of the normal inhibitory regulation of Na+ channels by CF transmembrane conductance regulator proteins.

Naturally Occurring Antisense Transcripts Are Present in Chick Embryo Chondrocytes Simultaneously with the Down-regulation of the α1(I) Collagen Gene

It has previously been shown that very low steady state levels of alpha 1 (I) collagen mRNA are present in chick embryo sternal chondrocytes (Askew, G. R., Wang, S., and Lukens, L. N. (1991) J. Biol. Chem. 266, 16834-16841), yet nuclear run-on experiments with double-stranded cDNA probes indicated a high transcription rate at this locus. These findings were investigated in this study using single-stranded probes, where nuclear run-on experiments showed that antisense transcription of the alpha 1 (I) collagen gene was occurring in chondrocytes, while sense strand transcription was down-regulated. Treatment of these chondrocytes with 5-bromo-2'-deoxyuridine (BrdU), which causes the cells to resemble their mesenchymal precursors, resulted in an antiparallel situation, where antisense transcription was lost, and instead, sense strand transcription was acquired, suggesting that the reverse switch from sense to antisense transcription occurs during chondrogenesis. Very large (> 10 kilobases) and heterogeneous antisense transcripts of moderate stability were shown to span both ends of the gene in chondrocytes, while their absence was shown in BrdU-treated chondrocytes, chick embryo fibroblasts, and a variety of other tissues. The function of these antisense transcripts is so far unknown, but their unusual chondrocyte-specific appearance, concurrent with little or no sense strand transcription, suggests a possible functional role in the down-regulation of the alpha 1 (I) collagen gene.

Insulin-like growth factor-I promotes cell proliferation in the absence of modulation of collagen phenotypes and utilizes IRS-1, not PLC-gamma 1, in corneal endothelial cells.

Corneal endothelial cells are differentiated cells and are thus incapable of physiologic regeneration. In a search for a growth factor that would promote optimal proliferation of corneal endothelial cells in the absence of other modulating activities, the effect of insulin-like growth factor-I (IGF-I) on rabbit corneal endothelial cells was studied. In addition, cellular effector molecules responsible for the signal pathway for IGF-I were studied. IGF-I at 50 ng/ml stimulated corneal endothelial cell proliferation after at least 8 h of treatment. IGF-I did not change cell shape of corneal endothelial cells: the cells treated with IGF-I at 50 ng/ml maintained polygonal morphology regardless of the duration of exposure. IGF-I did not alter collagen phenotypes either qualitatively or quantitatively: the treated cells continued to synthesize types IV and VIII collagen, as did the control cells. The steady-state levels of alpha 2(I) collagen RNA and alpha 2(IV) RNA were not altered by IGF-I treatment. Immunohistochemical analysis showed that IGF-I is present in corneal endothelium in vivo, while the underlying Descemet's membrane demonstrated no staining. Corneal endothelial cells also produce IGF binding protein-2 (IGFBP-2), which appears to bind IGF-I that has been introduced exogenously in the medium. Further investigation as to how the signals of IGF-I were transmitted for the biological activities demonstrated that the expression of insulin receptor substrate-1 (IRS-1) is up-regulated by IGF-I treatment, while PLC-gamma 1 expression is not altered by this growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental renal failure in the rat modulates cardiac Na,K-ATPase alpha 2 mRNA but not protein.

The decreased abundance and enzymatic activity of myocardial Na,K-ATPase have been recognized previously to occur in chronic uremia. However, the activity of the cardiac sodium pump as defined by the uptake of 86Rb is normal. The discrepancies between these findings may have resulted from the inability to distinguish between the different Na,K-ATPase isoforms now known to exist in cardiac muscle. To investigate this question, steady-state levels of Na,K-ATPase alpha and beta mRNA isoforms, alpha 1, alpha 2, and beta 1 protein, and specific high-affinity binding of [3H]ouabain were quantitated in cardiac muscle from uremic and pair-fed, sham-operated control rats. Steady-state levels of alpha 2 and beta 2 mRNA were significantly decreased (percentage of control levels: alpha 2, 48 +/- 10; beta 2, 74 +/- 9; N = 10; P < 0.025) in chronic renal failure without any change in alpha 1, alpha 3, or beta 1 expression. The number of high-affinity [3H]ouabain-binding sites and Na,K-ATPase alpha 1, alpha 2, and beta 1 subunits was not different from control. In acute renal failure, alpha 2 and beta 2 mRNA levels also were significantly decreased (percentage of control levels: alpha 2, 24 +/- 5; beta 2, 44 +/- 8; N = 6; P < 0.001), but there was no change in the level of alpha 3 or beta 1 mRNA, the number of high-affinity [3H]ouabain-binding sites, or the level of Na,K-ATPase alpha 2 and beta 1 subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs.

The steady-state level of alpha- and beta-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters alpha- and beta-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For beta-tubulin mRNAs, previous efforts have shown that this is the result of a selective mRNA degradation mechanism which involves cotranslational recognition of the nascent amino-terminal beta-tubulin tetrapeptide as it emerges from the ribosome. Site-directed mutagenesis is now used to determine that the minimal sequence requirement for conferring the full range of beta-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in alpha-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated alpha-tubulin genes reveals that alpha- and beta-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal alpha-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type alpha-tubulin transgenes nor an alpha-tubulin gene mutated to encode an amino-terminal MREI yields mRNAs that are autoregulated. Further, although slowing ribosome transit accelerates the autoregulated degradation of endogenous alpha- and beta-tubulin mRNAs, degradation of alpha-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, alpha- and beta-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.

Increases in type III collagen gene expression and protein synthesis in patients with inguinal hernias.

The aim of this study was to determine if alterations in fibrillar collagen synthesis were associated with the development of inguinal hernias. Previous work has suggested that alterations in connective tissue accumulation may play a functional role in the development of inguinal hernias. In particular, several investigators have suggested that alterations in collagen synthesis, causally related to connective disorders such as osteogenesis imperfecta, may also be responsible for the inguinal herniation that is markedly increased in such patients. This study was undertaken therefore to study collagen synthesis in patients with inguinal hernia in the absence of any other connective tissue disease. Skin fibroblasts from 9 patients with hernias and 15 control individuals were radiolabeled with 3H-proline. Trypsin-chymotrypsin-resistant type I and III collagens were isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and recovery was quantified by laser densitometry. Steady-state levels of alpha 1(I) and alpha 1(III) procollagen mRNAs were also determined by northern and slot-blot hybridization analysis. The alpha 1(I)/alpha 1(III) collagen ratios were shown to be 6.3 +/- 0.34 in fibroblasts from control individuals and 3.0 +/- 0.25 in fibroblasts from patients with inguinal hernias. This statistically significant difference (p < 0.0001) was caused by an increase in the secretion of alpha 1(III) procollagen from the fibroblasts of patients with hernias. A concomitant increase in the steady-state levels of alpha 1(III) procollagen mRNA was observed in total RNA isolated from the patients' fibroblasts. A constitutive and systemic increase in type III collagen synthesis may result in reduced collagen fibril assembly in the abdominal wall, eventually leading to the development of herniation. Although it is not yet clear what genetic factors are responsible for the elevation in type III collagen synthesis in patients with hernias, this study represents the first attempt to define individuals with an abnormality in collagen production that may be specifically related to herniation. A clearer understanding of the possible genetic factors that influence the pathophysiology of this disease will be important to improve the treatment of patients in whom inguinal hernias develop.

Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts

Dermal fibroblasts exposed to low oxygen tension show upregulated synthesis of transforming growth factor-beta 1 (TGF-beta 1), an established stimulatory peptide in the formation of extracellular matrix proteins. In this report, procollagen synthesis was measured in cultures of confluent adult human dermal fibroblasts exposed to either standard (20%) or low (2%) oxygen tension. By Northern blot analysis the steady state levels of alpha 1 (I) procollagen mRNA were increased by 75 to 150% of control (standard oxygen) as early as 12 hours and more than 200% 96 hours after exposure of cells to low oxygen. Similar increases in procollagen mRNA levels were obtained in hypoxic fibroblast cultures in a collagen lattice. The stimulatory effect of hypoxia on procollagen mRNA levels in fibroblast monolayers was diminished by antibodies to TGF-beta, and could not be augmented further by the addition of TGF-beta 1, evidence that hypoxic fibroblasts may already be maximally stimulated by TGF-beta 1. We conclude that low oxygen tension enhances steady state mRNA levels of alpha 1 (I) procollagen, and that this effect is mediated at least in part by TGF-beta 1.

Pleural mesothelial cell response to inflammation: tumor necrosis factor-induced mitogenesis and collagen synthesis.

This study examined the effects of tumor necrosis factor-alpha on pleural mesothelial cell proliferation and collagen synthesis, functions which may be important in the response of the pleura to injury. Tumor necrosis factor-alpha caused a significant increase in proliferation and collagen production by rat pleural mesothelial cells in vitro. Proliferation increased in a time- and dose-dependent manner, resulting in an approximate twofold increase in the uptake of [3H]thymidine relative to control. The uptake of [3H]proline into collagenase-sensitive protein increased in a dose-dependent manner for concentrations of tumor necrosis factor-alpha > or = 1.0 ng/ml. The increase in collagen production were associated with increased steady-state levels of alpha 1(I)-procollagen mRNA. These results suggest that tumor necrosis factor-alpha may have a significant effect on pleural mesothelial cell function in vivo in the setting of inflammation. Increases in pleural mesothelial cell proliferation and collagen synthesis in response to inflammatory mediators, like tumor necrosis factor-alpha, may be important in healing the pleura after injury by a variety of disease processes.

Expression of the gamma-subunit gene of desulfoviridin-type dissimilatory sulfite reductase and of the alpha- and beta-subunit genes is not coordinately regulated.

It has been shown [Pierik, A. J., Duyvis, M. G., van Helvoort, J. M. L. M., Wolbert, R. B. G. & Hagen, W. R. (1992) Eur. J. Biochem. 205, 111-115] that desulfoviridin, the dissimilatory sulfite reductase of sulfate-reducing bacteria of the genus Desulfovibrio, contains a third, gamma, subunit (11 kDa), in addition to the well-established alpha (50 kDa) and beta (40 kDa) subunits, and an alpha 2 beta 2 gamma 2 subunit structure has been proposed. Cloning and sequencing of the dsvC gene indicated it to encode a protein of 105 amino acids (11.9 kDa; gamma subunit). The finding that the dsvC gene, located on a 3.5-kb SacII fragment, is transcribed in both Escherichia coli and Desulfovibrio vulgaris as an mRNA of only 400-600 nucleotides, and that both the dsvA and dsvB genes are present on a 7.2-kb SacII fragment, indicates that dsvC forms a separate transcriptional unit. The steady-state level of alpha and beta subunits expressed in D. vulgaris Hildenborough cells is rather constant, while that of the gamma subunit increased strongly in the stationary growth phase. Biochemical analysis of the purified protein, expressed in E. coli, and library comparison of its sequence, have so far failed to establish the function of gamma.

Effects of platelet-derived growth factor isoforms on human lung fibroblast proliferation and procollagen gene expression.

Platelet-derived growth factor (PDGF), a potent mitogen for fibroblasts, is a potentially important cytokine in the pathogenesis of fibroproliferative disorders of lung. Different isoforms of PDGF include a heterodimer composed of A and B chains and homodimers composed of A or B chains. The biological significance of the different isoforms is unknown, but they have been shown to differ in their mitogenic potency in some systems. Their effects on other fibroblast functions have not been fully examined. We undertook this study to determine the effect of PDGF isoforms on human lung fibroblast proliferation and procollagen synthesis. Cultured lung fibroblasts (IMR-90, WI-38, GeNA) were incubated in the presence of varying concentrations of highly purified PDGF-AB obtained from platelets or recombinant PDGF-AA or -BB homodimers. Incorporation of [3H]thymidine was determined as a measure of mitogenic activity. Fetal lung fibroblasts (IMR-90, WI-38) and an adult fibroblast strain (GeNA) responded similarly to the different isoforms, with maximum mitogenic activity observed at 5-10 ng/mL. Cell cycle analysis using three additional normal adult lung fibroblast strains indicated that all PDGF isoforms stimulated similar proportions of cells to cycle over a 7-day period. Fibroblast procollagen synthesis, measured after pulse labeling with [3H]proline, was not increased even at concentrations of the PDGF isoforms that were maximally mitogenic. Moreover, steady-state levels of alpha 1(I) and alpha 1(III) procollagen mRNA levels, determined by northern analysis and dot blot hybridization, were not changed after exposure to any of the PDGF isoforms. While all PDGF isoforms are potent mitogens for fetal and adult lung fibroblasts, it was concluded that they do not directly stimulate procollagen gene expression or procollagen synthesis in vitro. The results suggest that PDGF isoforms are potentially important mitogens for lung fibroblasts, but other factors are likely to be involved in the stimulation of fibroblast procollagen synthesis that is observed in fibroproliferative disorders of lung.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

The interaction of heparin and basic fibroblast growth factor on collagen synthesis in 21-day fetal rat calvariae.

We examined the interactions of the glycosaminoglycan, heparin, and recombinant human basic fibroblast growth factor (bFGF) on collagen synthesis in 21-day fetal rat calvariae. In calvariae treated for 96 h, heparin (25 micrograms/ml) and bFGF (10(-9) M) inhibited collagenase-digestible protein (CDP) labeling by 52 and 60% of control, respectively, and the combination further inhibited CDP labeling. Inhibition of CDP labeling by heparin (25 micrograms/ml) or bFGF (10(-9), 10(-8) M) was similar in the presence or absence of aphidicolin (30 microM) an inhibitor of cell replication. Heparin selectively inhibited CDP labeling in the osteoblast rich central bone but bFGF alone or in combination with heparin inhibited CDP labeling both in the periosteum and central bone. Heparin and bFGF alone decreased steady state levels of alpha 1(I)procollagen messenger RNA (mRNA) at 24 h and the combination further decreased mRNA levels. A high concentration of insulin-like growth factor-1 (IGF-1, 3 x 10(-8) M) reversed the inhibitory effect of heparin on DNA synthesis and CDP labeling. In contrast, IGF-1 could not reverse the inhibitory effects of bFGF on CDP labeling but enhanced the stimulatory effects of bFGF on thymidine incorporation into DNA. We conclude that the inhibitory effects of heparin and bFGF on CDP are independent of effects on cell replication. We further conclude that both heparin and bFGF inhibit collagen synthesis at a pretranslational site since they decreased procollagen mRNA levels in osteoblasts. However, the inhibition of collagen synthesis by heparin and bFGF appears to involve divergent pathways since exogenous IGF-1 could overcome the effect of heparin but not bFGF on collagen synthesis.

Different levels of regulation accomplish the switch from type II to type I collagen gene expression in 5-bromo-2'-deoxyuridine-treated chondrocytes.

The shift of chick embryo chondrocytes to a fibroblastic phenotype by 5-bromo-2'-deoxyuridine (BrdUrd) has been used to examine the molecular basis of the switch from type II to type I collagen gene expression. Transcription rates of each of these three collagen genes before and after this shift were measured in nuclear run-on transcription assays with double-stranded 3'-cDNA probes specific for each of these three mRNAs. Degradation rates of each of these RNAs were calculated from the rate of decrease in the concentration of each RNA after the inhibition of synthesis with actinomycin D. The shut-off of the expression of the type II collagen gene during this shift was shown to occur at the transcriptional level, since the transcription rate of this gene decreased dramatically. The decay rate of the type II mRNA (half-life of approximately 15 h) is not significantly faster in BrdUrd-treated cells. The alpha 1(I) gene is transcribed at similar rates in untreated and shifted chondrocytes, but the steady state level of alpha 1(I) RNA in chondrocytes is only 1.5% of that in shifted cells. Although the measured degradation rate of the total alpha 1(I) RNA from untreated chondrocyte cultures is approximately the same as in shifted cells (half-life of approximately 12 h), indirect evidence suggests that this alpha 1(I) RNA is derived from a low level of fibroblast contamination of these chondrocyte cultures. The alpha 1(I) RNA synthesized by untreated chondrocytes is assumed therefore to be broken down very rapidly in the nucleus. The alpha 2(I) gene is also transcribed in untreated chondrocytes at rates similar to shifted cells but, unlike alpha 1(I) RNA, its steady state level in untreated chondrocytes is approximately 30% of its level in shifted chondrocytes. The increased level of alpha 2(I) RNA in shifted cells may be regulated in part by an increase in stability of the alpha 2(I) mRNA, which has half-lives of 5.2 and 10.4 h, respectively, in untreated and shifted chondrocytes. The alpha 2(I) RNA in the untreated chondrocytes was found to have a different 5' end from that present in the BrdUrd-shifted chondrocytes or in chick embryo fibroblasts. The presence of this altered RNA in untreated chondrocytes explains the absence of synthesis of the fibroblastic alpha 2(I) collagen polypeptide chains in these chondrocytes, despite the presence of the alpha 2(I) RNA as measured with 3' probes.

Stimulation of fibroblast collagen and total protein formation by an endothelial cell-derived factor.

We investigated the effect of medium conditioned by bovine aortic endothelial cells on collagen accumulation and total protein formation by human embryonic fibroblasts or bovine smooth muscle cells in cultures. The conditioned medium at a 1:10 dilution induced a twofold increase in collagen and total protein accumulation in fibroblast cultures. At low concentration (1:50 dilution), the conditioned medium stimulated collagen accumulation preferentially; at high concentration (1:10 dilution), overall protein synthesis also was increased. The increase in type I collagen accumulation was associated with an increase in the steady-state level of alpha 1 (I) mRNA for collagen. The conditioned medium increased the production of types I and III collagen without affecting the proportion of collagen types in both fibroblast and smooth muscle cell cultures. Partial purification of the endothelial cell-derived factor disclosed it to be a heat-stable protein with an apparent molecular weight of 8-10 kDa. The stimulation of protein formation by this substance was not inhibited by antibodies against transforming growth factor-beta or the insulinlike growth factor I receptor. The partially purified factor stimulated protein production without affecting fibroblast proliferation. This endothelial cell-derived protein may play a role in the remodeling of vascular connective tissue by stimulating collagen synthesis.

Differential degradation rates of the G protein alpha o in cultured cardiac and pituitary cells.

Signal transduction in biological membranes is modulated by a family of GTP-binding proteins termed G proteins. Differences in the tissue-specific expression of G protein subtypes suggest that the levels of individual G proteins may be an important determinant of the hormonal response in a given cell type. We have used a polyclonal antibody raised against the purified G protein, alpha o to study alpha o in the rat pituitary cell line GH4 and in primary rat cardiocytes in culture by quantitative immunoprecipitation. Biosynthetic labeling and specific immunoprecipitation of alpha o in pulse-chase experiments demonstrated that the t1/2 for alpha o degradation is 28 +/- 7 h (n = 4) in GH4 pituitary cells and is greater than 72 h (n = 4) in cardiocytes. The steady-state level of alpha o protein is similar in both cell types as measured by Western blots. Northern blots of poly(A)-selected mRNA from these two cell types were probed with labeled alpha o cDNA and showed they have similar alpha o mRNA levels. The observation of different degradation rates, but similar steady-state protein levels, suggests that the rate of alpha o synthesis is different in GH4 cells and cardiocytes. Since mRNA levels are approximately equal in both, our studies imply that protein translation controls may be important determinants of G protein alpha subunit concentrations in biological membranes.

Interferon beta 2/interleukin 6 modulates synthesis of alpha 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells.

The cytokine IFN beta 2/IL-6 has recently been shown to regulate the expression of genes encoding hepatic acute phase plasma proteins. INF beta 2/IL-6 has also been shown to be identical to MGI-2, a protein that induces differentiation of bone marrow precursor cells toward mature granulocytes and monocytes. Accordingly, we have examined the effect of IFN beta 2/IL-6 on expression of the IL-1- and tumor necrosis factor-unresponsive acute phase protein alpha 1-antitrypsin (alpha 1 AT) in human hepatoma-derived hepatocytes and in human mononuclear phagocytes. Purified human fibroblast and recombinant IFN beta 2/IL-6 each mediate a specific increase in steady-state levels of alpha 1 AT mRNA and a corresponding increase in net synthesis of alpha 1 AT in primary cultures of human peripheral blood monocytes as well as in HepG2 and Hep3B cells. Thus, the effect of IFN beta 2/IL-6 on alpha 1 AT gene expression in these cells is primarily due to an increase in accumulation of alpha 1 AT mRNA and can be distinguished from the direct, predominantly translational effect of bacterial lipopolysaccharide on expression of this gene in monocytes and macrophages. The results indicate that IFN beta 2/IL-6 regulates acute phase gene expression, specifically alpha 1 AT gene expression, in extrahepatic as well as hepatic cell types.

Gonadotrophin-releasing hormone regulation of gonadotrophin subunit gene expression: studies in tri-iodothyronine-suppressed rats

We have previously shown that a pulsatile gonadotrophin-releasing hormone (GnRH) stimulus can increase steady-state levels of alpha and LH-beta subunit mRNAs in the male rat pituitary. Since alpha subunit is produced in both thyrotroph and gonadotroph cells, the effect of GnRH specifically on gonadotroph alpha gene expression is uncertain. To address this tissue, adult male rats were given injections of tri-iodothyronine (T3; 20 micrograms/100 g body wt, i.p.) daily for 8 days (day 8 = day of death) in order to decrease thyrotroph alpha mRNA levels (+T3 group). Saline injections (i.p.) were given to control animals (-T3group). Three days before GnRH administration, the animals were castrated and testosterone implants inserted s.c., to inhibit endogenous GnRH secretion. GnRH pulses (25 ng/pulse; 30-min interval) were given to freely moving animals (saline pulses to controls) via an atrial cannula for 12,24 or 48 h. Serum LH and FSH were measured before and 20 min after the last GnRH pulse. Pituitary RNA was extracted and alpha, LH-beta, FSH-beta and prolactin mRNA levels were determined by dotblot hybridization using 32P-labelled cDNA probes. Castration and testosterone replacement reduced alpha and LH-beta mRNA levels by 30 and 40% respectively, compared with levels in untreated intact males, but did not decrease FSH-beta concentrations. T3 administration further decreased alpha mRNA to 30% of values seen in intact males, but LH-beta mRNA levels were unchanged. FSH-beta mRNA concentrations were decreased by 23% in T3-treated rats (P less than 0.05 vs intact controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of the rat α1(I) collagen promoter: Regulation by 1,25-dihydroxyvitamin D

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent reduction in steady state levels of alpha 1(I) and alpha 2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat alpha 1(I) collagen gene. This promoter fragment was fused to the chloramphenicol acetyltransferase gene and was introduced into ROS 17/2.8 cells by calcium phosphate co-precipitation. Expression of this construct was diminished by 1,25-(OH)2D3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibroblast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the alpha 1(I) promoter contains cis-active elements which are regulated by the 1,25-(OH)2D3 receptor in ROS 17/2.8 cells.