Expression of bovine myf5 induces ectopic skeletal muscle formation in transgenic mice.

myf5 is one of a family of four myogenic determination genes that control skeletal muscle differentiation. To study the role of myf5 in vivo, we generated transgenic mice harboring the bovine homolog, bmyf, under control of the murine sarcoma virus promoter. Ectopic expression of the full-length bmyf transgene was detected in brain and heart tissue samples of F1 progeny from transgenic founder mice. Ectopic bmyf expression activated endogenous skeletal myogenic determination genes in the hearts and brains of transgenic animals. Incomplete skeletal myogenesis in most hearts gave rise to cardiomegaly and focal areas of cardiomyopathy. In brains in which ectopic expression led to a more complete myogenesis, focal areas of multinucleated, striated myotubes containing actin, desmin, and myosin were observed. These unexpected results show that myf5 can initiate myogenic differentiation in vivo, supporting the hypothesis that myf5 is responsible for determination of cells to the myogenic lineage in normal embryogenesis.

Dynamic regulation of RGS2 in bone: potential new insights into parathyroid hormone signaling mechanisms.

The initial steps involved in mediating the transduction of PTH signal via its G protein-coupled receptors are well understood and occur through the activation of cAMP and phospholipase C pathways. However, the cellular and molecular mechanisms for subsequent receptor desensitization are less well understood. Recently, a new family of GTPase activating proteins known as regulators of G protein signaling (RGS), has been implicated in desensitization of several G protein-coupled ligand-induced processes. At present, it is not known whether any of the RGS proteins play a role in PTH signaling. Using the differential display method, we screened for genes that are selectively expressed after a single s.c. injection of human PTH (1-38) (8 microg/100 g) in osteoblast-enriched femoral metaphyseal spongiosa of young male rats (3-4 weeks old). We found and cloned one full-length complementary DNA that encodes a 211-amino acid RGS protein and shares 97% sequence identity with mouse and human RGS2. Based on sequence similarity, we have designated this clone as rat RGS2. Northern blot analysis confirmed that the expression of RGS2 messenger RNA (mRNA) is rapidly and transiently increased by human PTH (1-38) in both metaphyseal (4-to 5-fold) and diaphyseal (2- to 3-fold) bone, as well as in cultured osteoblast cultures (2- to 37-fold). In vitro, forskolin and dibutyryl cAMP similarly elevated RGS2 mRNA. In vivo, PTH analog (1-31) [which stimulates intracellular cAMP accumulation, PTHrP (1-34), and prostaglandin E2] induced RGS2 mRNA expression; whereas PTH analogs (3-34) and (7-34), which do not stimulate cAMP production, had no effect on expression. In tissue distribution analysis, RGS2 is widely expressed and was detected in all tissues examined (heart, spleen, liver, skeletal muscle, kidney, and testis), with significant expression in two nonclassical PTH-sensitive tissues: the brain, and the heart. After PTH injection, RGS2 mRNA expression was induced in rat bone but not in any of the other tissues examined. These findings demonstrate that RGS2 is regulated by PTH, prostaglandin E2, and PTHrP and that regulation by PTH in bone occurs via the cAMP pathway. Additionally, these results suggest the exciting possibility that increased RGS2 expression in osteoblasts may be one of the early events influencing PTH signaling.

ADAMTS-1: A cellular disintegrin and metalloprotease with thrombospondin motifs is a target for parathyroid hormone in bone.

PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single s.c. injection of human PTH-(1-38) (8 microg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1-38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1-31) and PTHrP-(1-34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3-34) and PTH-(7-34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE2 (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-kappaB ligand/transforming growth factor-beta1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE2, forskolin, or (Bu)2cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1alpha,25-dihydroxyvitamin D3 increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE2 possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.

Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells.

Novel bacterial resistance genes were cloned and expressed as dominant selection markers in mammalian cells. Escherichia coli genes coding for resistance to the aminocyclitol antibiotics hygromycin B (Hm) and G418 were cloned into the eukaryotic expression plasmid pSV5GPT [Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78 (1981) 2072-2076]. Mouse cells normally sensitive to 100 micrograms/ml Hm were transformed with these plasmids and selected in 200 micrograms/ml Hm. Transformants resistant to as much as 1 mg/ml Hm and 500 micrograms/ml G418 were isolated. Cell extracts contained an acetyltransferase activity capable of acetylating G418 and an Hm aminocyclitol phosphotransferase activity. Plasmid DNA sequences were identified by Southern blot analysis of high Mr DNA isolated from transformed cells.

Expression of potentially amyloidogenic derivatives of the Alzheimer amyloid precursor protein in cultured 293 cells.

The expression of the carboxyl-terminal 100 (C-100) residues of the amyloid precursor protein (APP) may provide a model for studying the processing of APP to the 42-43 residue beta-amyloid peptide (beta A4) implicated in Alzheimer's disease. Expression of human C-100 in mammalian cells reportedly causes 'toxicity' and amyloid-like fibrils. We have expressed the C-100 fragment in human embryonic kidney cells (293 cells) in a transient assay and compared it to the expression of transfected wild type and mutant (Swedish familial Alzheimer's disease) full length APP. Products were characterized by Western blot analysis using antibodies to the carboxyl-terminal region of APP.

Use of vectors to confer resistance to antibiotics g418 and hygromycin in stably transfected cell lines.

The development of dominant selection markers to identify eukaryotic cells that have undergone a gene transformation event has greatly facilitated molecular genetic studies in higher eukaryotic cells. Selection schemes based on resistance to antibiotic cytotoxicity (1,2) will be described in this chapter. Other schemes-for example, based on resistance to inhibition of DNA synthesis by methotrexate (1) or mycophenolic acid (1)-are described in other chapters of this book. Prior to the development of dominant selection markers, the use of recessive markers, such as thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyl transferase (HGPT) was limited to a handful of mutant cell lines that were TK (-) or HGPT(-) (5,6). If one wished to transfect a wild-type cell line, one had first to select a recessive mutant derivative cell line and characterize it before proceeding with the experiments of interest. Such restrictions posed a significant barrier to molecular genetic analyses in higher eukaryotic cells.

The determinants of hospital CEO compensation.

This study finds a significant difference in the compensation levels of public and private hospital CEOs. The difference, however, is not found to be associated with ownership structure. Rather, it is attributable to hospital characteristics such as size, location, and affiliation with a multihospital chain, and human characteristics such as gender, education, and experience.

Semi-quantitative fluorescence analysis of calcein binding as a measurement of in vitro mineralization.

We describe a new, highly sensitive semiquantitative method for rapid measurement of in vitro mineralization using calcein. Fluorescence analysis of the calcein bound to the calcium phosphate (hydroxyapatite) allows direct quantitation of extracellular matrix mineral content in monolayer cultures of bone-forming cells such as primary osteoblasts or osteosarcoma cells. Osteosarcoma cell lines UMR 106 and SaOS-2 were used to demonstrate that qualitatively, calcein was bound to the same regions of the mineralized cell monolayer as seen by conventional histological staining with von Kossa or Alizarin Red S. Moreover, total bound calcein could be quantitated by direct fluorescence analysis using a Cytofluor II plate reader. Changes in cell monolayer calcein fluorescence were shown to correlate well with direct colorimetric measurement of acid-solubilized Ca+2 from parallel cultures. Relative mineral quantitation by calcein fluorescence is rapid and more sensitive than colorimetric Ca+2 assays, can be performed directly on unfixed or fixed cell monolayers, and does not require the use of radioisotopes. The cell monolayer remains intact and potentially available for further analysis.

Hamster preproglucagon contains the sequence of glucagon and two related peptides.

Glucagon is a 29-amino acid polypeptide hormone synthesized by the A cells of the endocrine pancreas. Its primary site of action is the liver where it stimulates glycogenolysis, gluconeogenesis and ketogenesis. In mammals, biosynthetic studies have shown that glucagon is derived from a precursor of molecular weight (Mr) approximately 18,000 which is five to six times larger than glucagon. Glucagon-containing polypeptides and immunoreactants of various sizes have also been described from stomach, intestine, brain and salivary gland. Here, we have determined the structure of hamster pancreatic preproglucagon from the sequence of its cDNA. This 180-amino acid precursor contains the sequence of glucagon and two glucagon-like polypeptides arranged in tandem. The precursor also contains the sequences of several non-pancreatic glucagon-containing polypeptides which suggests that, in mammals, both pancreatic and non-pancreatic glucagon and glucagon-containing polypeptides may be derived from a common precursor by tissue-specific processing. We have tentatively identified each of the glucagon-like immunoreactants which have been described with respect to the sequence of proglucagon and have proposed a scheme for the processing of pancreatic proglucagon.

Single-stranded RNA probes generated from PCR-derived DNA templates.

The following report outlines the use of the polymerase chain reaction (PCR) in combination with in vitro transcription to generate single-stranded radiolabelled RNA probes useful for nuclease protection and in situ hybridization experiments. Specific DNA fragments with bacteriophage promoter (T3 and/or T7) sequences at the 5' or 3' end are generated by repeated rounds of amplification. Following purification, these PCR-generated DNA products are used as templates for in vitro transcription with the correct DNA-dependent RNA polymerase. The resultant radiolabelled, single-stranded RNA (ssRNA) can be used for in situ hybridization, Southern or Northern blot analysis, and ribonuclease protection experiments. Sub-cloning or hydrolysis of large fragments is not required. Probes can be made from virtually any sequence using a variety of template sources.

Temporal and quantitative analysis of myogenic regulatory and growth factor gene expression in the developing mouse embryo.

Using a reverse transcription/polymerase chain reaction method, the temporal pattern of expression of the myogenic regulatory genes (myf5, myogenin, MRF4, myo D) was quantitated in developing mouse muscle (whole embryo: 6.5 to 12.5 days postcoitum (dpc); front limb buds: 9.5 to 12.5 dpc; hind limb buds: 11.5 to 14.5 dpc) and related to expression of TGF-beta 1, b-FGF, IGF-I, and IGF-II. Myf5 was the first myogenic regulatory factor to appear in both the whole embryo and front limb bud, with expression evident 7.5 and 9.5 dpc, respectively. A transient peak of MRF4 expression occurred 10.5 dpc in both the whole embryo and the front limb bud. Myogenin and myo D expression in the whole embryo was detected 8.5 and 9.5 dpc, respectively. In the front limb bud myogenin and myo D expression was not detected until 10.5 dpc. In the hind limb bud myf5, myogenin, and MRF4 expression was detected 11.5 dpc. Myo D expression was not detected until 12.5 dpc. With respect to growth factor expression, in the front limb bud TGF-beta 1, IGF-I, and IGF-II were evident 9.5 dpc, while bFGF was not detected until 10.5 dpc. In the hind limb bud TGF-beta 1, bFGF, IGF-I, and IGF-II expression was detected 11.5 dpc. These results show that in both the whole embryo and limb buds, all four myogenic regulatory factors are involved in the initiation of the myogenic program. We also show myf5 expression in the 9.5-dpc front limb bud, suggesting its expression in the somite-derived migrating muscle precursor cells. Correlations between growth factor-mediated myoblast proliferation and myogenic differentiation are discussed.

Spontaneous atherosclerosis in pigeons. A model system for studying metabolic parameters associated with atherogenesis.

The interpretation of metabolic studies related to early changes in spontaneous atherosclerosis has been hampered by the focal nature of the disease and by the lack of a well-defined model system of the disease process. Gross, histologic and ultrastructural observations of lesion development at the celiac bifurcation of the aorta in atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons are compared and discussed in terms of hemodynamics, muscular aggregation and altered metabolism of smooth muscle cells. Detailed knowledge of the morphologic sequence of events in lesion localization makes the celiac bifurcation in White Carneau and Show Racer pigeons a useful model for genetic comparisons of arterial wall metabolism and for investigating metabolic alterations occurring with atherogenesis.

The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs.

Human liver cDNA coding for protein C has been synthesized, cloned and sequenced. The abundance of protein C message is approximately 0.02% of total mRNA. Three overlapping clones contain 1,798 nucleotides of contiguous sequence, which approximates the size of the protein's mRNA, based upon Northern hybridization. The cDNA sequence consists of 73 5'-noncoding bases, coding sequence for a 461 amino acid nascent polypeptide precursor, a TAA termination codon, 296 3'-noncoding bases, and a 38 base polyadenylation segment. The nascent protein consists of a 33 amino acid "signal", a 9 amino acid propeptide, a 155 amino acid "light" chain, a Lys-Arg connecting dipeptide, and a 262 amino acid "heavy" chain. Human protein C and Factor IX and X precursors possess about one third identical amino acids (59% in the gamma-carboxyglutamate domain), including two forty-six amino acid segments homologous to epidermal growth factor. Human protein C also has similar homology with prothrombin in the "leader", gamma-carboxyglutamate and serine protease domains, but lacks the two "kringle" domains found in prothrombin.

Immunohistochemical localization of selected early response genes expressed in trabecular bone of young rats given hPTH 1-34.

Intermittent administration of parathyroid hormone (PTH) increases trabecular bone mass in vivo by stimulating bone formation. To further characterize the cellular and molecular mediators of the anabolic response to PTH, we examined the effect of intermittent synthetic hPTH 1-34 on the expression and localization of selected early response genes, c-fos, c-jun, c-myc, and IL-6 protein, in bone tissue by immunohistochemistry. Young male Sprague-Dawley rats, 70-100 g, were injected s.c. with 8 microg/100 g PTH or vehicle control, once daily for 5 days. Femurs were harvested 1 and 24 hours after the fifth injection, then fixed, decalcified, processed for wax embedding, and sections were immunostained. Early response genes, c-fos, c-jun and IL-6, were strongly expressed in osteoblasts, osteocytes, and megakaryocytes in bones 1 hour after PTH, when compared with vehicle-treated controls or sections from rats, 24 hours after PTH injection. Osteoblasts, osteocytes, and megakaryocytes were also positive for c-myc but the differences in stain intensity between control and treated groups were marginal. Also, scattered islands of hematopoietic cells in the marrow stained intensely for IL-6 by 1 hour after PTH, but the stain intensity decreased to control level 24 hours after the last PTH injection. Scattered islands of hematopoietic cells in the bone marrow stained more strongly for c-fos than either c-jun or c-myc, but neither localization nor stain intensity were regulated by PTH at the time points examined. We conclude that during the immediate early phase of the anabolic response, PTH regulates c-fos, c-jun, and IL-6 expression in osteoblasts, osteocytes, megakaryocytes, and selected bone marrow hematopoietic cells in bone.

Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells.

A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 micrograms of insulin per mg of cell protein. [3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4/polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-1-methylxanthine. Insulin secretion at optimal glucose concentration (7.5 mM) was 2.4 milliunits per 10(6) cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis.

Molecular characterization of gene expression changes in ROS 17/2.8 cells cultured in diffusion chambers in vivo.

Transplantation of diffusion chambers (DC) containing osteoblast-like cells to extraskeletal sites has been highly studied and proven to be a useful technique to investigate the process of osteoblast differentiation and bone formation. To investigate the molecular basis of osteogenesis in DC, we examined the temporal pattern of gene expression of the proliferation marker histone H4, immediate early response genes (IEGs), c-fos, c-jun, c-myc, osteoblast phenotype-associated genes, osteocalcin (OC), osteopontin (OP), type I collagen (COL1A1), alkaline phosphatase (ALP), parathyroid hormone receptor (PTHR) and matrix modifying enzyme, matrix metalloproteinase-9 (MMP-9). DC containing ROS 17/2.8 were implanted intraperitoneally into rat hosts and cultured in vivo for various times up to 56 days. Histological analysis of von Kossa stained sections of the DC contents showed a well-organized connective tissue and the production of mineralized matrices/nodules. In contrast, histological examination of DC containing Rat-2 fibroblast cells revealed the lack of an organized mineralized matrix. Molecular analysis of DC containing ROS 17/2.8 cells at 0, 3, 10, 28, and 56 days demonstrated a time-dependent decrease in DNA content associated with cell death. In the surviving cells, an increase in histone H4 mRNA (consistent with an increase in cell proliferation) was evident by 3-10 days and thereafter expression returned to control levels. In vitro, ROS 17/2.8 cells expressed detectable levels of c-fos, c-jun, c-myc, OC, OP, ALP, COL1A1, and PTHR but not MMP-9. In vivo, the expression of c-fos increased 2-fold in 3-28 days and by 56 days was 4-5 fold above control levels. In 3-10 days, c-jun expression increased 1.6-1.8-fold above control levels. In contrast, by day 28, c-jun expression decreased to control levels, but increased to 2.1-fold above control by 56 days. c-myc mRNA expression increased 3-fold within 3 days and then dropped to below control values by 10-56 days. After transplantation in vivo, the expression of OC and PTHR decreased to undetectable levels. Similarly, ALP mRNA decreased to

Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization.

We report the novel synthesis of polymerase chain reaction (PCR)-derived single-stranded DNA (ssDNA) probes and their subsequent application in in situ hybridizations. Serial transverse sections of an 11.5-day postcoitum mouse embryo were hybridized to a 33P-ssDNA, 33P-RNA, or 35S-RNA probe corresponding to the same 181-bp sequence in the myogenin cDNA. Signal obtained using 33P-ssDNA was more intense than that using 33P-RNA probe, while signal/noise ratios obtained with both 33P-probes were far superior to those obtained with 35S-probe. Digoxigenin-labeled chicken growth hormone (GH) ssDNA gave slightly more intense signal than did digoxigenin-labeled chicken GH RNA when hybridized to chicken pituitary sections. 32P-ssDNA probes were found to be suitable for Northern blot hybridization. Advantages of using ssDNA probes for in situ hybridization include: (1) The ssDNA technique is rapid and simple. There was no need to clone a DNA template into a special RNA vector or order special T7-containing PCR primers. ssDNA probes can be synthesized in less than 1 day using any primers which currently exist in a laboratory (optimal probe length for in situ hybridization is between 50 and 200 bp). (2) In three separate in situ experiments, ssDNA probes yielded more intense signal than RNA probes. (3) ssDNA probes are potentially more stable than RNA probes. (4) Since the RNAse rinse is eliminated, posthybridization rinses are shortened when hybridizing with ssDNA probes. The ssDNA probes produced by this protocol can be labeled with a variety of different isotopes (both radioactive and nonradioactive), and are excellent probes for use in in situ hybridizations.

Nuclear and cytoplasmic localization of the beta-amyloid peptide (1-43) in transfected 293 cells.

Cultures of transformed human embryonic kidney 293 cells were transiently transfected with minigene constructs coding for the Abeta peptide (1-43). The Abeta minigene used in this study consisted of exons 16 and 17 of the amyloid precursor protein gene, including the 6000+ bp intronic region. Two of the constructs used in this study, human amyloid precursor protein (APP) promoter-driven Abeta minigene and BK virus enhancer/adenovirus major late promoter-driven Abeta minigene, did not contain a signal peptide sequence, whereas the third, human APP promoter-signal peptide Abeta minigene did not contain the human APP signal sequence. The resulting Abeta products were detected by immune precipitation, using 10D5 antibody and Western blot analysis, using R1280 antisera, as SDS stable oligomers in cell lysates of cells containing all three constructs or in culture media when produced by the signal peptide construct. Evaluation of the cells by immunocytochemistry using conventional and transmission electron microscopy indicated that the cells transfected with constructs without the signal peptide accumulated immunoreactive Abeta primarily in the nucleus.

Activation of osteocalcin transcription involves interaction of protein kinase A- and protein kinase C-dependent pathways.

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)- and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulin-like growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation of osteocalcin transcription is mediated by both PKA-dependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.