Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.     

Lung injury and mortality with hyperoxia are increased in peroxiredoxin 6 gene-targeted mice

Overexpression of peroxiredoxin 6 (Prdx6) has been shown to protect lungs of mice against hyperoxia-mediated injury. In this study, we evaluated whether genetic inactivation of Prdx6 in mice increases sensitivity to oxygen toxicity. We evaluated mouse survival, lung histopathology, total protein and nucleated cells in bronchoalveolar lavage fluid (BALF), and oxidation of lung protein and lipids by measurement of protein carbonyls and thiobarbituric reactive substances (TBARS), respectively. The duration of survival for Prdx6 -/- mice was significantly shorter than that observed in wild-type mice on exposure to 85 or 100% O(2); survival of Prdx6 +/- mice was intermediate. After 72-h exposure to 100% O(2), lungs of Prdx6-/- mice showed more severe injury than wild-type with increased wet/dry weight, epithelial cell necrosis and alveolar edema on microscopic examination, increased protein and nucleated cells in BALF, and higher content of TBARS and protein carbonyls in lung homogenate. These findings show that Prdx6 -/- mice have increased sensitivity to hyperoxia and provide in vivo evidence that Prdx6 is an important lung antioxidant enzyme.     

Lung phospholipid metabolism in transgenic mice overexpressing peroxiredoxin 6

Previous studies with peroxiredoxin 6 (Prdx6) null mice demonstrated that the phospholipase A(2) activity of this enzyme plays a major role in lung phospholipid metabolism. This study evaluated lung phospholipid metabolism in transgenic mice that over-express Prdx6. Lung lysosomal type PLA(2) activity in transgenic mice was 222% of wild type in lung homogenate and 280% in isolated lamellar bodies. Total phospholipid, phosphatidylcholine (PC) and disaturated PC were decreased approximately 20-35% in bronchoalveolar lung fluid, lung homogenate, and lung lamellar bodies in transgenic mice although lung compliance and type 2 cell ultrastructure were unaltered. To study metabolism, unilamellar liposomes ((3)H-DPPC: PC: cholesterol: PG, 10: 5: 3: 2 mol fraction) were instilled endotracheally in anesthetized mice and lungs were removed for perfusion. Compared to wild type, transgenic mice showed similar net uptake of liposomes in 2 h, but significantly increased (3)H-DPPC degradation (38.9+/-1.1 vs. 29.0+/-1.3% of recovered dpm). The PLA(2) competitive inhibitor MJ33 decreased degradation to 15% of recovered dpm in both transgenic and wild type lungs. Incorporation of [(14)C] palmitate into DSPC at 24 h after its intravenous injection was markedly increased in both the lung surfactant (+100%) and lamellar bodies (+188%) while incorporation of [(3)H] choline was increased by only 10-20%. These results indicate increased DPPC degradation and synthesis by the reacylation pathway with Prdx6 overexpression and provide additional evidence that the PLA(2) activity of Prdx6 has an important role in lung surfactant turnover.     

Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis

Growth hormone (GH) initiates its cellular action by properly dimerizing GH receptor (GHR). A substantial fraction of circulating GH is complexed with a high-affinity GH-binding protein (GHBP) that in many species can be generated by GHR proteolysis and shedding of the receptor's ligand-binding extracellular domain. We previously showed that this proteolysis 1) can be acutely promoted by the phorbol ester phorbol 12-myristate 13-acetate (PMA), 2) requires a metalloprotease activity, 3) generates both shed GHBP and a membrane-associated GHR transmembrane/cytoplasmic domain remnant, and 4) results in down-regulation of GHR abundance and GH signaling. Using cell culture model systems, we now explore the effects of GH treatment on inducible GHR proteolysis and GHBP shedding. In human IM-9 lymphocytes, which endogenously express GHRs, and in Chinese hamster ovary cells heterologously expressing wild-type or cytoplasmic domain internal deletion mutant rabbit GHRs, brief exposure to GH inhibited PMA-induced GHR proteolysis (receptor loss and remnant accumulation) by 60-93%. PMA-induced shedding of GHBP from Chinese hamster ovary transfectants was also inhibited by 70% in the presence of GH. The capacity of GH to inhibit inducible GHR cleavage did not rely on JAK2-dependent GH signaling, as evidenced by its continued protection in JAK2-deficient gamma2A rabbit GHR cells. The GH concentration dependence for inhibition of PMA-induced GHR proteolysis paralleled that for its promotion of receptor dimerization (as monitored by formation of GHR disulfide linkage). Unlike GH, the GH antagonist, G120K, which binds to but fails to properly dimerize GHRs, alone did not protect against PMA-induced GHR proteolysis; G120K did, however, antagonize the protective effect of GH. Our data suggest that GH inhibits PMA-induced GHR proteolysis and GHBP shedding by inducing GHR dimerization and that this effect does not appear to be related to GH site 1 binding, GHR internalization, or GHR signaling. The implications of these findings with regard to GH signaling and GHR down-regulation are discussed.     

Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism

Peroxiredoxin 6 (Prdx6), a bifunctional 25-kDa protein with both GSH peroxidase and phospholipase A2 activities, is the only mammalian 1-Cys member of the peroxiredoxin superfamily and is expressed in all major organs, with a particularly high level in lung. Prdx6 uses GSH as an electron donor to reduce H2O2 and other hydroperoxides including phospholipid hydroperoxides at approximately 5 micromol/mg protein/min with K1 approximately 3 x 10(6) M(-1) s(-1). Oxidation of the Cys47 to a sulfenic acid during catalysis requires piGST-catalyzed glutathionylation and reduction with GSH to complete the enzymatic cycle. Prdx6 stably overexpressed in cells protected against oxidative stress, whereas antisense treatment resulted in oxidant stress and apoptosis. Adenoviral-mediated overexpression of Prdx6 in mouse lungs protected against the toxicity of hyperoxia, whereas Prdx6-null mice were more sensitive to the effects of hyperoxia or paraquat. We postulate that Prdx6 functions in antioxidant defense mainly by facilitating repair of damaged cell membranes via reduction of peroxidized phospholipids. The PLA2 activity of Prdx6 is Ca2+ independent and maximal at acidic pH. Inhibition of PLA2 activity results in alterations of lung surfactant phospholipid synthesis and turnover. Thus, Prdx6, a unique mammalian peroxiredoxin, is an important antioxidant enzyme and has a major role in lung phospholipid metabolism.     

Regulation of full-length and truncated growth hormone (GH) receptor by GH in tissues of lit/lit or bovine GH transgenic mice

Two truncated isoforms of growth hormone (GH) receptor (GHR) were identified in mice and in humans. The proteins encoded by these isoforms lack most of the intracellular domain of the GHR and inhibit GH action in a dominant negative fashion. We have quantified the mRNAs encoding the GHR isoforms in mouse tissues by use of real-time RT-PCR and examined the effect of GH excess or deficiency on regulation of mRNA levels of the GHR isoforms in vivo. In the liver, the truncated GHR mRNAs (mGHR-282 and mGHR-280) were 0.5 and <0.1%, respectively, the level of full-length GHR (mGHR-fl). In skeletal muscle, the values were 2-3 and 0.1-0.5% of mGHR-fl, respectively, and in subcutaneous fat, the values were 3-5 and 0.1-0.5% of mGHR-fl, respectively. The bovine GH transgenic mice showed a significant increase of mGHR-fl in liver but a significant decrease in skeletal muscle, with no difference in subcutaneous fat when compared with control mice. The lit/lit mice showed a significant decrease of mGHR-fl in liver, no difference of mGHR-fl in muscle, and a significant increase of mGHR-fl in subcutaneous fat when compared with lit/+ mice. The mRNA of mGHR-282 was regulated in parallel with mGHR-fl in all tissues of all mice examined, whereas that of mGHR-280 was not changed in either GH-excess or GH-deficient states. In conclusion, two truncated isoforms of GHR mRNAs were detected in liver, skeletal muscle, and subcutaneous fat of mice. The ratio of GHR-tr to GHR-fl mRNA was tissue specific and not affected by chronic excess or deficiency of GH.     

Growth hormone action in the developing neural retina: a proteomic analysis

The possible presence and action of growth hormone (GH) in the neural retina was investigated in newborn mice. The neural retina was found to be a site of GH gene expression, as GH mRNA was abundant in cells of the retinal ganglion cell layer, in which GH was also detected. It was also a site of GH action, since GH receptor (GHR) immunoreactivity mirrored that of GH. Actions of GH within the eye were indicated by a reduction in its axial length and retinal width (its neuroblastic, inner plexiform, and optic fiber layers) in GHR gene disrupted mice (GHR-/-), in comparison with wild type (GHR+/+) littermates. In the absence of GH signaling, four proteins in the retinal proteome of the GHR-/- mice (identified by 2-D gels and MS) differed in abundance with those in the wild type mice. Brain abundant membrane attached signal protein-1 (BASP-1) was down-regulated, whereas protein kinase C inhibitor 1, cyclophilin A, KH domain-containing, RNA-binding, signal transduction-associated protein 3 were up-regulated in GHR-/- mice. These proteins are involved in retinal vascularization, neural proliferation and neurite outgrowth. GH might thus have hitherto unsuspected roles in these processes during retinal development.     

IGF-I deficiency due to GH receptor deficiency.

IGF-I deficiency may be primary due to defective synthesis, or secondary to GH receptor deficiency (GHRD) or defects in transduction of the GH-GHR signal. Cloning and sequencing of the GHR led to recognition that circulating GH binding protein (GHBP) was structurally identical to the extra-cellular domain of the GHR, and the identification of 33 mutations of the GHR in approximately half of the 250 patients that have been reported. This review explores the information provided about GHR function by various mutations, the population distribution of GHRD, the effects of this condition on mortality, growth, development, and metabolism, the effects of replacement therapy with recombinant human IGF-I, diagnostic issues, and the question of partial GH resistance.     

Platelet-derived growth factor and lysophosphatidic acid inhibit growth hormone binding and signaling via a protein kinase C-dependent pathway

Growth hormone (GH) regulates body growth and metabolism. GH exerts its biological action by stimulating JAK2, a GH receptor (GHR)-associated tyrosine kinase. Activated JAK2 phosphorylates itself and GHR, thus initiating multiple signaling pathways. In this work, we demonstrate that platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) down-regulate GH signaling via a protein kinase C (PKC)-dependent pathway. PDGF substantially reduces tyrosyl phosphorylation of JAK2 induced by GH but not interferon-gamma or leukemia inhibitory factor. PDGF, but not epidermal growth factor, decreases tyrosyl phosphorylation of GHR (by approximately 90%) and the amount of both total cellular GHR (by approximately 80%) and GH binding (by approximately 70%). The inhibitory effect of PDGF on GH-induced tyrosyl phosphorylation of JAK2 and GHR is abolished by depletion of 4beta-phorbol 12-myristate 13-acetate (PMA)-sensitive PKCs with chronic PMA treatment and is severely inhibited by GF109203X, an inhibitor of PKCs. In contrast, extracellular signal-regulated kinases 1 and 2 and phosphatidylinositol 3-kinase appear not to be involved in this inhibitory effect of PDGF. LPA, a known activator of PKC, also inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR and reduces the number of GHR. We propose that ligands that activate PKC, including PDGF, LPA, and PMA, down-regulate GH signaling by decreasing the number of cell surface GHR through promoting GHR internalization and degradation and/or cleavage of membrane GHR and release of the extracellular domain of GHR.     

Reduced proteolysis of rabbit growth hormone (GH) receptor substituted with mouse GH receptor cleavage site

GH binding protein (GHBP) is a circulating form of the GH receptor (GHR) extracellular domain, which derives by alternative splicing of the GHR gene (in mice and rats) and by metalloprotease-mediated GHR proteolysis with shedding of the extracellular domain as GHBP (in rabbits, humans, and other species). Inducible proteolysis of either mouse (m) or rabbit (rb) GHR is detected in cell culture in response to phorbol ester and other stimuli, yielding a cell-associated GHR remnant (comprised of the cytoplasmic and transmembrane domains and a small portion of the proximal extracellular domain) and down-regulating GH signaling. In this report, we map the mGHR cleavage site by adenoviral overexpression of a membrane-anchored mGHR mutant lacking its cytoplasmic domain and purification and N-terminal sequencing of the phorbol 12-myristate 13-acetate-induced remnant protein. The sequence obtained was LEACEEDI, which matches the mGHR extracellular domain stem region sequence L265EACEEDI272, indicating that mGHR cleavage occurs in the extracellular domain nine residues outside of the transmembrane domain, in the same region (but at different residues) as the rbGHR cleavage site we recently mapped. We studied the effects on receptor proteolysis and GHBP shedding of replacing rbGHR cleavage site residues with those corresponding to the mGHR cleavage site. We analyzed five separate rodentized rbGHR mutants incorporating mGHR amino acids either at or surrounding the cleavage site. Each mutant was normally processed, displayed at the cell surface, and responded to GH stimulation by undergoing tyrosine phosphorylation. Only the mutants replaced with mGHR cleavage site residues, rather than surrounding residues, exhibited deficient inducible proteolysis and GHBP shedding. These findings suggested that the GHR cleavage sites in the two species differ in their susceptibility to cleavage. This difference may underlie interspecies variation in utilization of proteolysis to generate GHBP.     

Surfactant protein A regulates surfactant phospholipid clearance after LPS-induced injury in vivo

Previous in vitro studies have suggested that surfactant protein A (SP-A) may play a role in pulmonary surfactant homeostasis by mediating surfactant secretion and clearance. However, mice made deficient in SP-A [SP-A (-/-) animals] have relatively normal levels of surfactant compared with wild-type SP-A (+/+) animals. We hypothesize that SP-A may play a role in surfactant homeostasis after acute lung injury. Bacterial lipopolysaccharide was instilled into the lungs of SP-A (-/-) mice and SP-A (+/+) mice to induce injury. Surfactant phospholipid levels were increased 1.6-fold in injured SP-A (-/-) animals, although injury did not alter [3H]choline or [14C]palmitate incorporation into dipalmitoylphosphatidylcholine (DPPC), suggesting no change in surfactant synthesis/secretion 12 h after injury. Clearance of [3H]DPPC from the lungs of injured SP-A (-/-) animals was decreased by approximately 40%. Instillation of 50 microg of exogenous SP-A rescued both the clearance defect and the increased phospholipid defect in injured SP-A (-/-) animals, suggesting that SP-A may play a role in regulating clearance of surfactant phospholipids after acute lung injury.     

Critical role of peroxiredoxin 6 in the repair of peroxidized cell membranes following oxidative stress

Phospholipids are a major structural component of all cell membranes; their peroxidation represents a severe threat to cellular integrity and their repair is important to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A₂ (PLA₂) activity, plays a critical role in antioxidant defense of the lung and other organs. We investigated the role of Prdx6 in the repair of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVEC) and isolated mouse lungs treated with tert-butyl hydroperoxide and lungs from mice exposed to hyperoxia (100% O₂). Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances, oxidation of diphenyl-1-pyrenylphosphine, or ferrous xylenol orange assay. The exposure dose was varied to give a similar degree of lipid peroxidation at the end of exposure in the different models. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild-type PMVEC and perfused lungs but were unchanged in Pxdx6 null preparations. An intermediate degree of repair was observed with PMVEC and lungs that expressed only C47S or D140A mutant Prdx6; the former mutant does not have peroxidase activity, while the latter loses its PLA₂ activity. Prdx6 null mice showed markedly delayed recovery from lipid peroxidation during 20 h observation following exposure to hyperoxia. Thus, Prdx6 plays a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress; the peroxidase and PLA₂ activity each contribute to the recovery process.     

Isolation, characterization, and distribution of two cDNAs encoding for growth hormone receptor in rainbow trout (Oncorhynchus mykiss)

Growth hormone (GH) plays important roles in a vast array of physiological processes, including growth, metabolism, and reproduction. In this study, cDNAs for two unique growth hormone receptor variants were cloned and sequenced from rainbow trout. The two cDNAs, one consisting of 2920 bp and the other of 2820 bp, share 87.2% identity in nucleotide sequence and 85.5% identity in deduced amino acid sequence and presumably arose through gene duplication. The cDNAs encode for putative 593- and 594-amino acid growth hormone receptors (designated GHR1 and GHR2, respectively), each containing a single transmembrane domain and other motifs characteristic of the receptor family. Both GHR1 and GHR2 mRNAs were present in all tissues examined. Trout GHR mRNAs are differentially expressed, both in terms of abundance among tissues and in terms of abundance within selected tissues. GHR1 was more abundant than GHR2 in the brain, whereas GHR2 was more abundant than GHR1 in pancreas and spleen. These findings expand our understanding of the evolution of the GH receptor family and suggest that independent mechanisms serve to regulate the tissue-specific expression of GHR mRNAs.