Downstream target genes of the neuropeptide S-NPSR1 pathway.

The neuropeptide S (NPS)-NPS receptor 1 (NPSR1) pathway has recently been implicated in the pathogenesis of asthma. The purpose of this study was to identify downstream gene targets regulated by NPSR1 upon NPS stimulation. A total of 104 genes were found significantly up-regulated and 42 down-regulated by microarray analysis 6 h after NPS administration. By Gene Ontology enrichment analysis, the categories 'cell proliferation', 'morphogenesis' and 'immune response' were among the most altered. A TMM microarray database comparison suggested a common co-regulated pathway, which includes JUN/FOS oncogene homologs, early growth response genes, nuclear receptor subfamily 4 members and dual specificity phosphatases. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), exhibited a significant NPS dose-response relationship as confirmed by quantitative reverse-transcriptase-PCR and for MMP10 by immunoassay. Immunohistochemical analyses revealed that MMP10 and TIMP metallopeptidase inhibitor 3 (TIMP3) were both strongly expressed in bronchial epithelium, and macrophages and eosinophils expressed MMP10 in asthmatic sputum samples. Because remodeling of airway epithelium is a feature of chronic asthma, the up-regulation of MMP10 and TIMP3 by NPS-NPSR1 signaling may be of relevance in the pathogenesis of asthma.

Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris.

High-level recombinant expression systems for the production of stable triple-helical human collagens and collagen fragments have been developed in the yeast Pichia pastoris. Collagen fragments are secreted as single-chain polypeptides by the yeast alpha-mating factor pre-pro sequence, but secretion of full-length triple-helical procollagen molecules has not been achieved despite the use of the same secretory signal. We studied here the effects of the secretory signal and the conformation and size of the collagen polypeptide on its secretion in P. pastoris. Unlike the collagen signal sequence, the alpha-mating factor pre-pro sequence led to efficient secretion of single-chain 45 and 9 kDa type I collagen fragments. The efficiency was dependent on the length of the collagen polypeptide, as secretion of single-chain full-length 90 kDa alpha1(I) polypeptides was less efficient than that of the 45 kDa fragment. Furthermore, the conformation of the collagen polypeptides had a marked effect on secretion, as induction of trimerization of the 45 and 9 kDa fragments by either the C propeptide or the small trimerizing domain foldon led to an accumulation of triple-helical molecules inside the cells despite the presence of the alpha-mating factor pre-pro sequence. Our results show that P. pastoris is a suitable host for the development of tailored expression systems aimed at selective production of nonsecreted triple-helical and secreted single-chain collagen fragments of varying lengths for specific purposes.

Characterization of GPRA, a novel G protein-coupled receptor related to asthma.

We recently identified a novel positional asthma susceptibility gene, GPRA, which belongs to the G protein-coupled receptor family. In the present studies, we show that isoform specific activation of GPRA-A with its agonist, Neuropeptide S (NPS) resulted in significant inhibition of cell growth. GPRA has several variants due to extensive alternative splicing. We observed that only the full-length variants, GPRA-A and GPRA-B, with 7 transmembrane topology are transported into the plasma membrane, while the truncated proteins retain intracellular compartments. To clarify disease mechanism, we studied co-expression of the variants without finding any indication that truncated variants would inhibit the receptor transport into the plasma membrane. By using in situ hybridization and immunohistochemistry, we detected ubiquitous expression of GPRA-B, and frequent expression of GPRA-A in the epithelia of several organs including bronchi and gastrointestinal tract. Furthermore, we observed aberrant mRNA and protein expression levels of GPRA in the asthmatic bronchi. Finally, we demonstrate that GPRA and NPS are co-expressed in bronchial epithelium. In summary, this study provides evidence that GPRA might have functional relevance in modulating asthma by increased expression levels in the relevant tissues under diseased state and by potential inhibitory effect of GPRA-A activation on cell growth.

Characterization of a common susceptibility locus for asthma-related traits.

Susceptibility to asthma depends on variation at an unknown number of genetic loci. To identify susceptibility genes on chromosome 7p, we adopted a hierarchical genotyping design, leading to the identification of a 133-kilobase risk-conferring segment containing two genes. One of these coded for an orphan G protein-coupled receptor named GPRA (G protein-coupled receptor for asthma susceptibility), which showed distinct distribution of protein isoforms between bronchial biopsies from healthy and asthmatic individuals. In three cohorts from Finland and Canada, single nucleotide polymorphism-tagged haplotypes associated with high serum immunoglobulin E or asthma. The murine ortholog of GPRA was up-regulated in a mouse model of ovalbumin-induced inflammation. Together, these data implicate GPRA in the pathogenesis of atopy and asthma.

Involvement of proteasome in the dynamic assembly of the androgen receptor transcription complex.

We have used the chromatin immunoprecipitation technique to analyze the formation of the androgen receptor (AR) transcription complex onto prostate-specific antigen (PSA) and kallikrein 2 promoters in LNCaP cells. Our results show that loading of holo-AR and recruitment of RNA polymerase II to the promoters occur transiently. The cyclic nature of AR transcription complex assembly is also illustrated by transient association of coactivators GRIP1 and CREB-binding protein and acetylated histone H3 with the PSA promoter. Treatment of cells with the pure antiandrogen bicalutamide also elicits occupancy of the promoter by AR. In contrast to the agonist-liganded AR, bicalutamide-bound receptor is not capable of recruiting polymerase II, GRIP1, or CREB-binding protein, indicating that the conformation of AR bound to anti-androgen is not competent to assemble transcription complexes. Proteasome is involved in the regulation of AR-dependent transcription, as a proteasome inhibitor, MG-132, prevents the release of the receptor from the PSA promoter, and it also blocks the androgen-induced PSA mRNA accumulation. Furthermore, occupancy of the PSA promoter by the 19 S proteasome subcomplex parallels that by AR. Collectively, formation of the AR transcription complex, encompassing AR, polymerase II, and coactivators, on a regulated promoter is a cyclic process involving proteasome function.

Characterization of three fragments that constitute the monomers of the human lysyl hydroxylase isoenzymes 1-3. The 30-kDa N-terminal fragment is not required for lysyl hydroxylase activity.

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80-85 kDa, corresponding to three fragments A-C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B-C polypeptide constituted a fully active hydroxylase with K(m) values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä, R. (2000) J. Biol. Chem. 275, 36158-36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo.