Promoter regulatory elements and DNase I-hypersensitive sites involved in serglycin proteoglycan gene expression in human erythroleukemia, CHRF 288-11, and HL-60 cells.

We have compared regulation of the serglycin gene in human erythroleukemia (HEL) and CHRF 288-11 cells, which have megakaryocytic characteristics, with promyelocytic HL-60 cells. Deletion constructs were prepared from the region -1123/+42 to -20/+42, and putative regulatory sites were mutated. In all three cell lines, the two major regulatory elements for constitutive expression were the (-80)ets site and the cyclic AMP response element (CRE) half-site at -70. A protein from HEL and CHRF, but not HL60, nuclear extracts bound to the (-80)ets site. Another protein from all three cell lines bound to the (-70)CRE. Phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (dbcAMP) increased expression of the reporter in HEL cells 2.5-3- and 4.5-fold, respectively, from all constructs except those with (-70)CRE mutations. PMA virtually eliminated expression of serglycin mRNA and promoter constructs, but dbcAMP increased expression in HL-60 cells. The effects of PMA and dbcAMP on promoter expression correlated with mRNA expression. The strengths of two DNase I-hypersensitive sites in the 5'-flanking region and the first intron in all three cells correlated with relative endogenous serglycin mRNA expression. An additional DNase I-hypersensitive site in HL60 DNA in the first intron may be related to the high serglycin expression in HL60 relative to HEL or CHRF cells.

Serglycin proteoglycan synthesis in the murine uterine decidua and early embryo.

This study has explored the localization and synthesis of the serglycin proteoglycan in the murine embryo and uterine decidua during midgestation. Embryos in deciduae were subjected to in situ hybridization with cRNA probes and to immunohistochemical detection with a specific antibody against murine serglycin. Adherent decidual cell cultures were prepared from freshly isolated deciduae. Proteoglycan biosynthesis was investigated by labeling intact deciduae and decidual cultures with (35)S-sulfate. Serglycin mRNA was detected by in situ hybridization throughout the mesometrial portion and at the periphery of the antimesometrial portion of the decidua at Embryonic Day (E) 8.5, and in the parietal endoderm surrounding the embryo. Serglycin mRNA was detected in fetal liver at E11.5-E14.5. Serglycin was detected by immunohistochemistry in decidua and parietal endoderm at E8.5 and in liver at E13.5. Most of the proteoglycans synthesized by cultured intact deciduae (78%) and adherent decidual cultures (91%) were secreted into the medium. Serglycin proteoglycan may play an important role in uterine decidual function during early postimplantation development.

Mechanisms of proliferation synergy by receptor tyrosine kinase and G protein-coupled receptor activation in human airway smooth muscle.

Despite recent studies depicting the capacity of G protein-coupled receptors (GPCRs) to activate mitogenic signaling pathways more commonly associated with receptor tyrosine kinases (RTKs), little is known regarding the interactive effects of GPCR and RTK activation on cell growth and signal transduction. Such interactions likely mediate the physiologic growth in most cells in vivo as well as the aberrant, non-neoplastic growth that occurs in diseases such as asthma, where disruptions of the local hormonal or inflammatory state can contribute to significant GPCR activation. In this study, we show that numerous inflammatory or contractile agents, including thrombin, histamine, and carbachol, potentiate epidermal growth factor (EGF)-stimulated proliferation of human airway smooth muscle (ASM), thus demonstrating a clear synergy between RTK and GPCR activation. Alterations in promitogenic nuclear signaling were evidenced by additive or synergistic increases in Elk-1 and activator protein-1 activation, and by increases in cyclin D1 expression. Interestingly, GPCR activation did not cause EGF receptor tyrosine phosphorylation nor did it increase EGF-stimulated autophosphorylation. In the presence of EGF, histamine or carbachol did not alter the time-dependent phosphorylation of p42/p44, whereas thrombin was capable of increasing phospho-p42/p44 levels at selected time points in some, but not all, cultures. In contrast to their relative inability to alter EGF receptor-linked p42/p44 activation, thrombin, histamine, and carbachol consistently increased the late phase (> 1 h) activity of p70 S6 kinase. Collectively, these findings suggest that inflammatory and contractile agents that activate GPCRs can significantly modulate RTK-mediated ASM growth through a p70 S6 kinase-dependent, p42/p44-independent mechanism.