Expression of a parathyroid hormone-like protein and its receptor during differentiation of embryonal carcinoma cells.

Differentiation of mouse embryonal carcinoma cells to the parietal endoderm phenotype is associated with expression of PTH-responsive adenylate cyclase. A PTH-like protein (PLP), which binds to PTH receptors and activates adenylate cyclase in classical PTH target cells was recently isolated and cloned. We assessed whether the parietal endoderm phenotype is associated with the expression of PLP or its receptor. A 1.4-kilobase PLP transcript was detected in the mouse parietal endoderm cell line PYS-2. No hybridizing transcripts were evident in undifferentiated mouse embryonal carcinoma cells PSA-1 or F9. However, differentiation of these cells to parietal endoderm, either spontaneously (PSA-1) or by treatment with retinoic acid and dibutyryl cAMP (F9), resulted in expression of the 1.4-kilobase PLP message. Undifferentiated F9 cells displayed negligible specific binding of [125I]PLP-(1-34)amide. When F9 cells were induced to differentiate to parietal endoderm, specific binding sites for [125I]PLP-(1-34)amide were expressed in parallel with PLP-responsive adenylate cyclase. These receptors, like those in classical PTH target tissues, displayed identical affinity (Kd = 5.2 nM) for bPTH-(1-34) and hPLP-(1-34)amide; with binding capacity (Bmax) of 6.6 x 10(4) sites/cell. In the presence of retinoic acid, exogenous PLP substituted for dibutyryl cAMP in a concentration-dependent fashion in promoting the differentiation of F9 cells to parietal endoderm. Thus, both PLP mRNA and PLP receptors coupled to adenylate cyclase are expressed during the differentiation of mouse embryonal carcinoma cells. Increased cAMP levels produced by autocrine stimulation of PLP receptors by PLP may contribute to differentiation of embryonal carcinoma cells into parietal endoderm.

Chicken parathyroid hormone-related protein and its expression during embryologic development.

A parathyroid hormone-related protein (PTHrP) is the probable cause of humoral hypercalcemia in malignancy, but its normal physiologic role remains unknown. Since current evidence suggests that PTHrP may have a role in embryonic development, we cloned a genomic fragment that encodes chicken PTHrP (cPTHrP) and studied the expression of PTHrP in developing chick embryos. Blot hybridization of chicken genomic DNA with a cPTHrP genomic DNA probe showed a single band, suggesting that a single-copy gene encodes cPTHrP. By screening a chicken genomic library with the human probe an open reading frame was identified that corresponds to the human PTHrP (hPTHrP) exon IV. Compared to the human sequence the 5' splice junction is highly conserved and the two predicted propeptide residues are identical. The sequence predicts a mature peptide of 139 amino acids; all of the first 21 and 94 of the first 112, but only 8 of the final 27 residues of cPTHrP are identical to the human sequence. The structural features required for PTH receptor binding and activation are highly conserved between chicken and hPTHrP. Poly(A)-enriched RNA from 3-15 day chicken embryos was surveyed by hybridization to the chicken probe. A hybridizing band of 1.45 kb was found in tissues derived from all three germ layers, including brain, heart, lung, liver, gizzard, intestine, chorioallantoic membrane, yolk sac, and skeletal muscle. An additional 1.2 kb hybridizing band was found in some tissues. The conservation of the PTHrP sequence between chicken and mammals supports the view that PTHrP has an important physiologic role.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of the regulation of the 1 alpha,25-dihydroxyvitamin D3 receptor in the rat jejunum by glucocorticoids.

The distribution of 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptors in isolated jejunal villous and crypt cells was investigated in normal and adrenalectomized male rats, and also in animals treated with the synthetic glucocorticoid, dexamethasone, and/or the glucocorticoid antagonist, 11-deoxy-cortisol. Adrenalectomy caused an increase in 1,25-(OH)2D3 receptors whilst dexamethasone treatment led to a reduction in receptor number. 11-Deoxy-cortisol was able to reverse the 'down-regulation' effect caused by glucocorticoids. In all cases, the changes in receptor numbers were more pronounced in crypt cells. The data suggest that, in the small intestine, glucocorticoids may control the synthesis of 1,25-(OH)2D3 receptors via the mediation of a glucocorticoid receptor, and that the adrenal hormones mainly express their effect in crypt cells. It is proposed that this phenomenon may, in part, explain the reduction in calcium absorption which occurs in man after chronic glucocorticoid treatment.

Detection of plasmids using DNA and RNA probes and the light-addressable potentiometric sensor.

Intact plasmids, plasmid fragments, and cDNA were detected using two DNA or RNA probes of varying lengths, each containing only biotin or fluorescein molecules. The probes were hybridized with the target plasmid/cDNA, bound with streptavidin, captured on nitrocellulose membranes, and detected using the urease-conjugate of an anti-fluorescein antibody via the light-addressable potentiometric sensor. The output of the silicon-chip sensor is in the form of rate, microV/s, and is directly proportional to the quantity of hybridized DNA captured on the membrane. Use of the larger probes (for beta-actin cDNA target) results in detection of intact plasmid at the level of approximately 106 target molecules; complementary DNA could be detected at similar levels. When the smaller probes are utilized (pGEM or pBSActB targets), plasmid fragmented targets could be detected only at levels 200 times greater than those observed when using the larger RNA probes.

Logos: the emblem in the marketing wars.

The logo is a powerful marketing tool. It is a condensed form of communication for a selected niche in a mass audience. Effective logo design principles include considerations of art design, visual perception, memory retention, and succinct nonverbal communication. Design principles for logos for dental practices are presented. Dental practices may realize significant advantages if logos are properly conceived and executed and are creatively implemented.

Distribution and properties of Ca2+-ATPase, phytase, and alkaline phosphatase in isolated enterocytes from normal and vitamin D-deficient rats.

The effects of vitamin D-deficiency and repletion on the distribution and activities of Ca2+-ATPase, phytase, and alkaline phosphatase in intact epithelial cells isolated from different regions of the villi and the crypts of the rat jejunum were studied. Similar distribution patterns of activities were found for the three enzymes. In all cases, the enzyme levels were the highest at the villus tip and gradually declined to low activities in the crypt. The Kms were very different between cells in the crypt base and those at the villus tip, the highest Kms being found in the crypt. The activities of these enzymes were reduced in the entire length of the villus in vitamin D-deficient rats. Recovery of the enzymatic levels was observed on vitamin D repletion, but at different rates. Total recovery of activity of Ca2+-ATPase, phytase, and alkaline phosphatase was observed after 18, 24, and 36 hours, respectively, after a single dose of 6.5 nmol (2.5 micrograms) vitamin D3. Enzymatic activities in the crypt cells were not affected by vitamin D3 treatment. These data suggest that Ca2+-ATPase, phytase, and alkaline phosphatase may be distinct entities, and that their activities in the crypt cells may not be vitamin D-dependent.

The temporal distribution of the 1 alpha,25-dihydroxycholecalciferol receptor in the rat jejunal villus.

The distribution of the 1 alpha,25-dihydroxycholecalciferol receptor was studied in enterocytes isolated from the upper, mid and lower villus and crypt cells of the jejunum of normal and rachitic rats. In all cell fractions a high-affinity receptor (KD congruent to 0.07 nmol/l) with a sedimentation coefficient of 3.5S was demonstrated. In normal rats there was a 60% reduction in receptor numbers in crypt cells compared with the mid and upper villous cells. Vitamin D deficiency led to a reduction in receptor numbers in all cell fractions (45% upper villus, 78% crypt cells). The data are compatible with the concept of calcium absorption occurring in the differentiated villous cells and also account for the reduction in absorption in rachitic animals.

Oophorectomy leads to a selective decrease in 1,25-dihydroxycholecalciferol receptors in rat jejunal villous cells.

The distribution of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] receptors in isolated rat jejunal cells was investigated in normal and oophorectomized female rats and also in vitamin D-deficient animals. Oophorectomy caused a selective reduction in 1,25-(OH)2D3 receptor numbers in villous cells, the reduction being similar (37%) in both normal and vitamin D-deficient animals. The data suggest that this effect of oophorectomy reflects a direct effect of oestrogens in the intestinal synthesis of 1,25-(OH)2D3 receptors, independent of the renal synthesis of 1,25-(OH)2D3. It is proposed that this phenomenon may, in part, explain the reduction in calcium absorption which occurs after oophorectomy or the menopause.

Transcriptional activation of Gs alpha expression by retinoic acid and parathyroid hormone-related protein in F9 teratocarcinoma cells.

Cyclic AMP is known to enhance retinoic acid-induced differentiation of F9 mouse teratocarcinoma cells to parietal endoderm. Recently, we showed that a parathyroid hormone-related protein (PTHrP), by activating adenylate cyclase, can substitute for exogenous cAMP in promoting retinoic acid-induced differentiation of F9 cells. However, the mechanisms by which endogenous cAMP levels are regulated during F9 differentiation are poorly defined. We therefore assessed whether Gs alpha, a subunit of the stimulatory coupling protein of adenylate cyclase, is induced during this process. Treatment of F9 cells with retinoic acid (1 microM) for 5 days resulted in a 20-fold increase in steady-state levels of a 2.0-kilobase Gs alpha mRNA. This was accompanied by an increase in the expression of 52- and 45-kDa Gs alpha polypeptides. Gs alpha mRNA increases within 24 h of exposure to retinoic acid, whereas the expression of alpha 1 (IV) collagen, a marker for F9 differentiation, did not increase until 48 h of treatment. In the presence of retinoic acid, exogenous human PTHrP-(1-34)-amide (20 nM) produced a further 2-fold increase in Gs alpha mRNA. These effects of retinoic acid and PTHrP were exerted largely if not entirely at the level of Gs alpha gene transcription, as assessed by nuclear run-on assay. Bt2cAMP (1 mM) did not reproduce the stimulatory effects of PTHrP on Gs alpha transcription, mRNA, or protein. These data demonstrate that a marked increase in Gs alpha expression accompanies F9 differentiation induced by retinoic acid and PTHrP, and that the regulation is predominantly transcriptional. The resulting increase in adenylate cyclase responsiveness to PTHrP and perhaps other ligands may be a critical component of the differentiation process. The effect of PTHrP on the expression of Gs alpha appears to be mediated by signals other than cAMP.

Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors.

G-protein-coupled receptors are a major target for potential therapeutics; yet, a large number of these receptors couple to the Gi pathway, generating signals that are difficult to detect. We have combined chimeric G proteins, automated sample handling, and simultaneous 96-well fluorometric imaging to develop a high-throughput assay system for Gi signaling. The chimeric G proteins alter receptor coupling so that signaling can occur through Gq and result in mobilization of intracellular calcium stores. An automated signaling assay device, the fluorometric imaging plate reader (FLIPR), can simultaneously measure this response in real time in 96-well microplates, allowing two people to process more than 10,000 points per day. We used the chimeric G protein/FLIPR system to characterize signaling by the Gi-coupled human opioid receptors. We show that the mu, delta, and kappa opioid receptors and the related nociceptin receptor, ORL1, each couple to Galphaqi5, Galphaqo5, and Galpha16 (Galphaqi5 and Galphaqo5 refer to Galphaq proteins containing the five carboxyl-terminal amino acids from Galphai and Galphao, respectively) and that different receptor/G protein combinations show different levels of maximal activation. We tested 31 opioid ligands for agonist activity at the opioid receptors (124 ligand-receptor combinations); all 31 activated at least one receptor type, and several activated multiple receptors with differing potencies. This high-throughput assay could be useful for dissecting the complex ligand-receptor relationships that are common in nature.

Nonisotopic quantitation of mRNA using a novel RNase protection assay: measurement of erbB-2 mRNA in tumor cell lines.

We have developed a nonisotopic RNase protection assay using RNA probes that are dual-labeled with biotin and fluorescein for detection. This system utilizes capture of the protected RNA probe hybrids to streptavidin-coated membranes attached to plastic dipsticks, complexing of anti-fluorescein-urease conjugate with the labeled RNA probe, and quantitative detection of the membrane-bound complex by a potentiometric silicon sensor. The dual-label RNase protection (RP) assay was capable of measuring beta-actin mRNA in cellular RNA samples at the 27- to 45-amol level (10-17 pg) with high precision (%CV < 7). We have used this method to quantitate the levels of erbB-2 mRNA in the human tumor cell lines SKBR-3, SKOV-3, and MCF-7. The levels of erbB-2 mRNA in these cells were 105, 190, and 0.9 amol per microgram of cellular RNA, respectively. The dual-label RP method should be useful for measuring the mRNA expression for other erbB-2 homologs such as erbB-3 and erbB-4 in tumor cells and tissues and can be a generally useful mRNA quantitative method for laboratories wishing to minimize radioisotope use.

The gene for the alpha 2 chain of the human fibrillar collagen type XI (COL11A2) assigned to the short arm of chromosome 6.

A cosmid clone (CosHcol.11) containing the alpha 2(XI) collagen gene (COL11A2) has been isolated. The gene contains conserved DNA and amino-acid sequences characteristic of fibril forming collagen, which is in accordance with the classification of type XI collagen as a fibrillar collagen. The genomic clone containing the alpha 2(XI) gene has been used as probe in the Southern blot analysis of DNA from a panel of human/hamster somatic cell hybrids containing different numbers and combinations of human chromosomes. Synteny analysis revealed that only chromosome 6 showed complete concordant segregation with COL11A2. Furthermore, the gene was regionally mapped to the short arm of chromosome 6 by using a hybrid which contained only the long arm of the chromosome.

Controlling signaling with a specifically designed Gi-coupled receptor.

We are developing a system to control G protein signaling in vivo to regulate a broad range of physiologic responses. Our system utilizes G protein-coupled peptide receptors engineered to respond exclusively to synthetic small molecule ligands and not to their natural ligand(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). We have made two prototype RASSLs that are based on the human kappa opioid receptor. Small molecule drugs that activate the kappa receptor are nonaddictive and safe to administer in vivo. Binding and signaling assays reveal 200-2000-fold reductions in the ability of our RASSLs to bind or be activated by dynorphin, an endogenous peptide ligand of the kappa opioid receptor. In a high-throughput signaling assay, these prototype RASSLs expressed in Chinese hamster ovary K1 cells showed little or no response to a panel of 21 opioid peptides but still signaled normally in response to small molecule drugs such as spiradoline. Activation of a RASSL by spiradoline also caused proliferation of rat-1a tissue culture cells. These data provide evidence that G protein-coupled receptors can be made into RASSLs. The potential in vivo applications for RASSLs include the positive enrichment of transfected cells and the development of new animal models of disease.

Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.

HER2, the erbB-2/neu proto-oncogene product, is a 185-kDa transmembrane glycoprotein related to the epidermal growth factor receptor. Overexpression of HER2 was reported in several human adenocarcinomas, including mammary and ovarian carcinomas. A family of glycoproteins, the heregulin/neu differentiation factors, was characterized and implicated as the ligands for HER2. Recently, it has been shown that HER2 alone is not sufficient to reconstitute high affinity heregulin receptors and that HER3 or HER4 may be the required components of the heregulin receptors on mammary carcinoma cells (Sliwkowski, M.X., Schaefer, G., Akita, R.W., Lofgren, J.A., Fitzpatrick, V.D., Nuijens, A., Fendly, B.M., Cerione, R.A., Vandlen, R.L., and Carraway, K.L., III (1994) J. Biol. Chem. 269, 14661-14665; Plowman, G.D., Green, J.M., Culouscou, J.-M., Carlton, G.W., Rothwell, V.M., and Buckley, W. (1993) Nature 366, 473-475). Using the Cytosensor to measure the extracellular acidification rate, we have examined the effects of recombinant human heregulin-alpha on three mammary carcinoma cell lines expressing HER2 (MDA-MB-453, SK-BR-3, and MCF-7), an ovarian carcinoma cell line expressing HER2 (SK-OV-3), and CHO-K1 and 293-EBNA cells stably transfected with HER2. By reverse transcription polymerase chain reaction and Western blotting, we found that the breast cells also express HER3 and that the ovarian line co-expresses the HER4 message. A dramatic increase in the acidification rate was observed for the mammary carcinoma cells co-expressing high levels of HER2 and HER3. In contrast, the ovarian cells expressing high levels of HER2 and low levels of HER4 or CHO-K1 and 293-EBNA cells expressing HER2 alone were not responsive to heregulin. When these same transfected cells were exposed to monoclonal anti-HER2 antibody followed by anti-IgG to cause aggregation of the HER2 molecules, an increase in the acidification rate was observed, indicating coupling of transfected HER2 to the signal transduction pathway. Transfection of HER2 into MCF-7 cells, on the other hand, gave 4-fold enhanced acidification responses. These data, together with the previously reported high affinity heregulin binding and activation of tyrosine phosphorylation in HER2 and HER3 co-transfected cells support the role of HER2 and HER3 as components of the heregulin receptor in breast cells.

Duration and magnitude of nerve growth factor signaling depend on the ratio of p75LNTR to TrkA.

The role of the low affinity neurotrophin receptor p75LNTR in neurotrophin signal transduction remains open. Recent reports show that this receptor generates intracellular signals independent of Trk activity, and others imply that it collaborates with Trk(s) to enhance cellular responses to low neurotrophin concentrations. We have used the Cytosensor microphysiometer as a direct marker of intracellular metabolic activity to address the physiologic role of p75LNTR in nerve growth factor (NGF) signal transduction. NGF treatment of PC12 or TrkA-transfected Chinese hamster ovary (CHO) cells results in a rapid, transient increase in the extracellular acidification rate as measured by the Cytosensor; in both cell types, p75LNTR enhances this response. p75LNTR affects both the magnitude of and the duration of the extracellular acidification response to NGF. Moreover, it is not merely the presence of p75LNTR, but also the ratio of p75LNTR:TrkA which determines cellular responsiveness to NGF. In transiently transfected CHO cells, a 5:1 ratio of p75LNTR:trkA cDNAs produced the greatest change in NGF-induced acid secretion. Pretreatment of PC12 cells with anti-p75LNTR antibodies decreased the responsiveness to NGF. However, long-term NGF exposure to PC12 cells in which p75LNTR expression was decreased to approximately 10% of wild-type levels showed a longer duration of acid secretion compared to wild-type PC12 cells. Together, these data suggest that p75LNTR may play a dual role in modulating NGF signal transduction by enhancing and extending cellular responses to short-term ligand exposures while attenuating the metabolic response to long-term ligand exposures. With regard to potential Trk-independent p75LNTR signal transduction mechanisms, we detected no change in extracellular acidification response in 75LNTR-transfected CHO cells, PCNA-15 fibroblasts, or Schwann cells, all of which express large amounts of p75LNTR and no Trk. Thus, p75LNTR cannot produce any signal detected by microphysiometry in the absence of TrkA.

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization.

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.

Two homologs of the Drosophila polarity gene frizzled (fz) are widely expressed in mammalian tissues.

The frizzled (fz) locus of Drosophila encodes a protein (Fz) with a seven-transmembrane-domain profile characteristic of G-protein-coupled receptors. In Drosophila, genetic evidence suggests that Fz functions to transmit and transduce polarity signals in epidermal cells during hair and bristle development. We have isolated from a UMR 106 rat osteosarcoma cell library a cDNA (fz-1) encoding a predicted 641-residue protein (Fz-1) with 46% homology with Drosophila Fz. We also identified a second cDNA (fz-2) encoding a protein (Fz-2) of 570 amino acids that is 80% homologous with Fz-1, with divergence most evident in the extracellular domains. Southern blots of rat genomic DNA indicated that fz-1 and fz-2 represent distinct genes. Northern analysis revealed the presence of a single fz-1 mRNA (4.7 kilobases) and two fz-2 mRNAs (2.5 and 4.5 kilobases) in rat tissues. The fz-1 and fz-2 genes are widely expressed in rat tissues with the highest steady-state levels of mRNA in kidney, liver, heart, uterus, and ovary. fz-1 and -2 mRNA levels were greater in neonatal than in corresponding adult tissues. Treatment of UMR 106 cells with bone resorbing agents including parathyroid hormone, epidermal growth factor, and 1,25-dihydroxyvitamin D3 produced increases in fz-1 and -2 mRNA levels. We suggest that hormonal induction of Fz proteins in osteoblasts serves to promote intercellular signaling required for functional responses such as increased bone resorption. Fz-1 and Fz-2 may represent products of a gene family whose members serve as transducers or intercellular transmitters of signals required for normal morphogenesis and/or differentiated function in diverse tissues.