Cloning of a cDNA encoding a novel cytochrome P450 from the insect Locusta migratoria: CYP6H1, a putative ecdysone 20-hydroxylase.

The biosynthesis of the steroidal molting hormone, 20-hydroxyecdysone, of arthropods involves a series of cytochrome P450-catalyzed hydroxylations. None of the many sequences of insect cytochromes P450, known to date, is related to ecdysteroid pathways. Here, we report the cloning and sequencing of a full-length cDNA of a new cytochrome P450, classified as CYP6H1, from malpighian tubules of the locust, Locusta migratoria. The 1854 bp DNA contained an open reading frame coding for a protein of 542 amino acids, a 5'-leader sequence and a 3'-untranslated region containing a polyadenylation signal and a poly(A) tail. The encoded protein had been isolated as an ecdysone-binding cytochrome P450 from microsomes of the same tissue in previous work. The closest homolog of CYP6H1 was CYP6A2 from Drosophila with 42.1% identity. According to Northern analysis, CYP6H1 is predominantly expressed at larval instars and in malpighian tubules. Evidence is presented for a functional assignment of CYP6H1 to microsomal ecdysone 20-hydroxylase of the locust.

Formoterol and isoproterenol induce c-fos gene expression in osteoblast-like cells by activating beta2-adrenergic receptors.

Formoterol, a beta2-adrenergic agonist has been shown in ovariectomized rat models to have anabolic effects on bone. However, those studies did not determine whether the effect of formoterol was by a direct action on bone cells themselves or indirectly via anabolic action on muscle. To address the question of whether formoterol could directly affect osteoblast function we investigated the expression patterns of beta3-adrenergic receptors (betaARs) in human osteoblast-like cells and functional coupling to gene expression. Northern blot analysis showed that betaAR subtypes are expressed at different levels in the osteoblast-like cell lines TE-85, SaOS-2, MG-63, and OHS-4. beta1AR expression was found in SaOS-2, OHS-4, and TE-85, but not MG-63 cells. beta2ARs are expressed at higher levels in MG-63 cells than in TE-85 and SaOS-2 cells, but were not detected in OHS-4 cells. PCR analysis paralleled the northern blot analysis except that beta3AR expression was found in one of three human primary osteoblast cDNAs tested. beta3AR expression was not found in any of the osteoblast-like cell lines. The nonspecific betaAR agonist, isoproterenol, and the beta2AR-specific agonist, formoterol, induced c-fos gene expression in cultured SaOS-2 cells in an immediate early fashion. This effect was inhibited by the beta2AR-specific antagonist, ICI 118551, but not by the beta1AR-specific antagonist, CGP 20712, indicating that induction of c-fos gene expression is specifically mediated by beta2ARs. c-fos gene expression was induced by both isoproterenol and formoterol via increases in cAMP, which in turn activated the cAMP/PKA pathway; the PKA inhibitor, H89, inhibited c-fos gene expression. Thus, betaARs are expressed in osteoblast-like cells and are coupled to c-fos gene expression via the beta2AR, increases in cAMP levels and activation of a PKA-dependent pathway.

The ubiquitous VA68 isoform of subunit A of the vacuolar H(+)-ATPase is highly expressed in human osteoclasts.

The 67-kDa subunit A of the vacuolar-type H(+)-ATPase carries the high affinity ATP binding site and together with the 57-kDa subunit B forms the catalytic domain. Two isoforms of subunit A, VA68 and HO68, were cloned from an osteoclastoma cDNA library. We have analyzed their respective expression patterns in different tissues by RNAase A protection and in situ hybridization. The HO68 isoform was found to be present only in the tumor originally used to construct the cDNA library, whereas the ubiquitous VA68 RNA isoform was detected in large osteoclastic cells, as well as in brian and kidney, by RNAse protection assay. Furthermore, we localized the strong signal observed in osteoclastoma RNA to the large osteoclastic cell by in situ hybridisation. These findings suggest that the subunit A highly expressed in human osteoclasts is the ubiquitous isoform, VA68.

Heterogeneity of vacuolar H(+)-ATPase: differential expression of two human subunit B isoforms.

The catalytic domain of the vacuolar proton ATPase is composed of a hexamer of three A subunits and three B subunits. Here we describe the cloning and characterization of a cDNA isoform of subunit B, HO57, from an osteoclastoma cDNA library. HO57 is represented by three species of mRNA of 1.6, 2.6 and 2.8 kb and is expressed at low levels in a range of human tissues, but at significantly higher levels in brain, kidney and osteoclastoma, and is probably an ubiquitously expressed isoform. In contrast, the kidney-specific isoform has an mRNA of 2 kb and is specifically expressed at high levels only in kidney and, at a lower level, in placenta. Thus the HO57 isoform is integral to the vacuolar ATPase found in the general secretory system of all cells as well as in vacuolar-ATPase-rich sources such as neurones and osteoclasts, whereas both the kidney-specific isoform and HO57 are highly expressed in the kidney. Furthermore, we show by in situ hybridization that HO57 is the only isoform that is exclusively and highly expressed by osteoclasts.

Cloning and tissue distribution of subunits C, D, and E of the human vacuolar H(+)-ATPase.

The vacuolar proton ATPase (V-ATPase) translocates protons into intracellular organelles or across the plasma membrane of specialised cells such as osteoclast and renal intercalated cells. The catalytic site of the V-ATPase consists of a hexamer of three A subunits and three B subunits which bind and hydrolyse ATP and are regulated by accessory subunits C, D and E. cDNAs encoding subunits C, D, and E were cloned from human osteoclastoma, a tissue highly enriched in osteoclasts, as a first step in the characterisation of the V-ATPase used by the osteoclast. By Northern blot analysis only one mRNA species were detected for each of these subunits, which is consistent the constant transcription level in all tissues irrespective of the presence of specialised cells highly enriched in V-ATPases.

Identification of two subunit A isoforms of the vacuolar H(+)-ATPase in human osteoclastoma.

Subunit A is thought to be the main component of the catalytic site of the vacuolar-type H(+)-ATPase. Screening of a cDNA library made from human osteoclastoma tumor tissue revealed the presence of two isoforms of subunit A. HO68 is a cDNA of 3.1 kilobase pairs, corresponding to a mRNA of approximately 3.4 kilobases in osteoclastoma only, encoding a protein of 615 amino acids with a predicted molecular mass of 68177 Da. A second subtype, VA68, corresponding to a mRNA of approximately 4.8 kilobases was present in all tissues analyzed, and codes for a predicted protein of 617 residues and theoretical molecular mass of 68264 Da. These clones share homology with previously published subunit A sequences, and this, together with the tissue distribution of the mRNA, suggests there are ubiquitous (VA68-type) and tissue-specific (HO68-type) isoforms. HO68 shows the closest sequence homology (95% at the amino acid level) to subunit A of a proton-secreting vacuolar-type H(+)-ATPase located in the apical membrane of midgut goblet cells of tobacco hornworm larva (Manduca sexta). We propose that HO68 could correspond to an isoform of subunit A specific for a vacuolar-type H(+)-ATPase located in the osteoclast plasma membrane.

Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin's disease.

We have identified a cDNA coding for a protein of 160 kDa which is expressed in in vitro cultured human peripheral blood monocytes. The predicted amino acid sequence contains an alpha-helical rod domain possessing features characteristic of intermediate filament proteins. However, the immunocytochemical staining pattern, abundance and solubility in Triton X-100/high salt buffers suggest that this protein is probably only associated with the intermediate filament network and represents a new type of intermediate filament associated protein. In a survey of normal, inflammatory and human tumour tissue samples, this protein, which we have named restin, was found to be highly expressed in Reed-Sternberg cells, the tumoral cells diagnostic for Hodgkin's disease. We suggest that restin overexpression may be a contributing factor in the progression of Hodgkin's disease.