Chronic hypoxia elevates intracellular pH and activates Na+/H+ exchange in pulmonary arterial smooth muscle cells

Chronic hypoxia (CH), caused by many lung diseases, results in pulmonary hypertension due, in part, to increased muscularity of small pulmonary vessels. Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to growth factors requires increased intracellular pH (pHi) mediated by activation of Na+/H+ exchange (NHE); however, the effect of CH on PASMC pHi homeostasis is unknown. Thus we measured basal pHi and NHE activity and expression in PASMCs isolated from mice exposed to normoxia or CH (3 wk/10% O2). pHi was measured using the pH-sensitive fluorescent dye BCECF-AM. NHE activity was determined from Na+-dependent recovery from NH4-induced acidosis, and NHE expression was determined by RT-PCR and immunoblot. PASMCs from chronically hypoxic mice exhibited elevated basal pHi and increased NHE activity. NHE1 was the predominate isoform present in mouse PASMCs, and both gene and protein expression of NHE1 was increased following exposure to CH. Our findings indicate that exposure to CH caused increased pHi, NHE activity, and NHE1 expression, changes that may contribute to the development of pulmonary hypertension, in part, via pH-dependent induction of PASMC proliferation.     

Structural basis of ion pumping by Ca(2+)-ATPase of sarcoplasmic reticulum

The structures of the Ca(2+)-ATPase (SERCA1a) have been determined for five different states by X-ray crystallography. Detailed comparison of the structures in the Ca(2+)-bound form and unbound (but thapsigargin-bound) form reveals that very large rearrangements of the transmembrane helices take place accompanying Ca2+ dissociation and binding and that they are mechanically linked with equally large movements of the cytoplasmic domains. The meanings of the rearrangements of the transmembrane helices and those of the cytoplasmic domains, and the mechanistic roles of the phosphorylation are now becoming clear.     

A proteomics approach to identify proliferating cell nuclear antigen (PCNA)-binding proteins in human cell lysates. Identification of the human CHL12/RFCs2-5 complex as a novel PCNA-binding protein

Proliferating cell nuclear antigen (PCNA), a eukaryotic DNA replication factor, functions not only as a processivity factor for DNA polymerase delta but also as a binding partner for multiple other factors. To understand its broad significance, we have carried out systematic studies of PCNA-binding proteins by a combination of affinity chromatography and mass spectrometric analyses. We detected more than 20 specific protein bands of various intensities in fractions bound to PCNA-fixed resin from human cell lysates and determined their peptide sequences by liquid chromatography and tandem mass spectrometry. A search with human protein data bases identified 12 reported PCNA-binding proteins from both cytoplasmic (S100 lysate) and nuclear extracts with substantial significance and four more solely from the nuclear preparation. CHL12, a factor involved in checkpoint response and chromosome cohesion, was a novel example found in both lysates. Further studies with recombinant proteins demonstrated that CHL12 and small subunits of replication factor C form a complex that interacts with PCNA.     

Early placental insulin-like protein (INSL4 or EPIL) in placental and fetal membrane growth

Early placental insulin-like protein (INSL4 or EPIL) is a member of the insulin superfamily of hormones, which is highly expressed in the placenta. We have confirmed this at term and shown it to be expressed by the maternal decidua. Although an abundance of locally acting growth factors are produced within the uterus during pregnancy, we hypothesized that INSL4 plays an important role in fetal and placental growth. We have demonstrated with cell lines and primary cells that it has a growth-inhibitory effect by causing apoptosis and loss of cell viability. We used primary amniotic epithelial cells for flow cytometry to show that INSL4 caused apoptosis, which was dose-related and significant (P < 0.05) at 50 ng/ml. This was confirmed by measurement of the nuclear matrix protein in the media. In comparison, relaxin treatment (up to 200 ng/ml) had no effect on apoptosis. The addition of INSL4 (3-30 ng/ml) also caused a loss of cell viability, although it had no effect on the numbers of cells at different phases of the cell cycle. Placental apoptosis is an important process in both normal placental development and in fetal growth restriction. Therefore, an in vivo clinical correlate was sought in fraternal twins exhibiting discordant growth. Expression of the INSL4 gene was doubled in the placenta of the growth-restricted twin compared to the normally grown sibling, suggesting that it may be linked to a higher level of apoptosis and loss of cell viability and, therefore, that it may contribute to fetal growth restriction.     

ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan

ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including Delta Sp (Delta Arg(693)-Lys(837), lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC(50) = 110 nm), whereas no inhibition was observed with Delta Sp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC(50) = 170 nm). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.     

Tyrosine kinase 2 interacts with and phosphorylates receptor for activated C kinase-1, a WD motif-containing protein

Receptor for activated C kinase (Rack)-1 is a protein kinase C-interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase Cgamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alphabeta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudokinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N terminus and one in the middle portion (aa 138-203) of Rack-1. Jak activation causes the phosphorylation of tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.     

Involvement of EAT/mcl-1, an anti-apoptotic bcl-2-related gene, in murine embryogenesis and human development

Apoptosis plays an important regulatory role in mammalian embryogenesis and development. EAT/mcl-1 (EAT), an anti-apoptotic bcl-2-related gene, was isolated during the early differentiation of a human embryonal carcinoma cell line, an event which serves as a model of early embryogenesis. EAT is involved in apoptotic regulation and is believed to also function as an immediate-early gene. Thus it was hypothesized that EAT would be expressed during early embryogenesis and would be involved in the regulation of apoptosis during this critical period. To clarify this early expression, two antibodies to EAT were generated by use of immunizing oligopeptide (aa 37-55) and recombinant protein (aa 31-229) for use in immunohistochemistry and immunoblotting, respectively. With these antibodies, we then determined EAT expression during murine embryogenesis and in human development, using human fetal tissue of 6 to 23 gestational weeks. During murine embryogenesis, the EAT protein was found to be rapidly induced after fertilization, to peak at the 2-cell stage, to remain constant until the 8-cell stage, and then to decrease to below unfertilized egg levels in blastocysts. EAT expression patterns in early human development were found to essentially overlap those observed in adult tissues which suggest that EAT expression continues until adulthood in terminally differentiated tissues. Among tissues distinct to fetal development, EAT was detected in the mesonephric (Wolffian) duct and paramesonephric (Müllerian) duct. It is also noteworthy that prominent EAT immunoreactivity was also observed in large primary oocytes in 21-week fetal ovary, but was not detected in primordial germ cells in 23-week fetal testis. In summary, EAT expression was detected in hematopoietic, epithelial, neural, endocrine, and urogenital cells; this provides evidence that EAT, as an anti-apoptotic molecule, possibly functions to regulate apoptosis during development in these systems.     

Morphological evidence for neural interactions between leptin and orexin in the hypothalamus

Both leptin and orexin have been recently discovered as peptides involved in feeding regulation. The morphological evidence of neural interaction between leptin and orexin, one considered to inhibit food intake and the other to stimulate it in the central nervous system (CNS), was studied by use of double immunostaining method. The leptin receptor-like immunoreactive (LR-LI) neurons in the hypothalamic arcuate nucleus and ventromedial nucleus were innervated by orexin-like immunoreactive (OX-LI) neurons. The distribution of LR-LI neurons in the hypothalamus was very similar to that of OX-LI neurons. These results may suggest that leptin and orexin are intimately correlated with each other and that they reciprocally regulate feeding at the hypothalamic level.     

Pitavastatin enhanced BMP-2 and osteocalcin expression by inhibition of Rho-associated kinase in human osteoblasts

To clarify the mechanism of the stimulatory effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on bone formation, we investigated the effect of pitavastatin, a newly developed statin, on expression of bone morphogenetic protein-2 (BMP-2) and osteocalcin in primary cultured human osteoblasts. Pitavastatin increased the expression level of mRNA for BMP-2, and much more effectively for osteocalcin. This stimulatory effect was abolished by the addition of geranylgeranyl pyrophosphate, an essential molecule for prenylation of small GTP-binding proteins such as Rho GTPase, but not by inhibitors of nitric oxide synthase and various protein kinases. Pitavastatin suppressed the Rho-associated kinase (Rho-kinase) activity. Hydroxyfasudil, a specific inhibitor of Rho-kinase, increased BMP-2 and osteocalcin expression. These mRNA levels were strongly suppressed by dexamethasone, but restored by co-treatment with hydroxyfasudil. These observations suggest that the Rho-kinase negatively regulates bone formation and the inhibition of Rho and Rho-kinase pathway is the major mechanism of the statin effect on bone. Moreover, a Rho-kinase inhibitor may be a new therapeutic reagent for the treatment of osteoporosis such as glucocorticoid-induced osteoporosis.