Lysyl oxidase is associated with increased thrombosis and platelet reactivity.

Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Lox(tg/tg)) were generated. Pf4-Lox(tg/tg) mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox(tg/tg) mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Lox(tg/tg) platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox(tg/tg) platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2ß1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.

Expression of a phosphorylated substrate domain of p130Cas promotes PyMT-induced c-Src-dependent murine breast cancer progression.

Elevated expression of p130Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. p130Cas is a downstream target of the tyrosine kinase c-Src. Signaling mediated by p130Cas through its phosphorylated substrate domain (SD) and interaction with effector molecules directly promotes tumor progression. We previously developed a constitutively phosphorylated p130Cas SD molecule, Src*/SD (formerly referred to as Src*/CasSD), which acts as decoy molecule and attenuates the transformed phenotype in v-crk-transformed murine fibroblasts and human breast cancer cells. To test the function of this molecule in vivo, we established mouse mammary tumor virus (MMTV)-long terminal repeat-Src*/SD transgenic mice in which mammary gland development and tumor formation were analyzed. Transgenic expression of the Src*/SD molecule under the MMTV-long terminal repeat promoter did not interfere with normal mammary gland development or induce tumors in mice observed for up to 11 months. To evaluate the effects of the Src*/SD molecule on tumor development in vivo, we utilized the MMTV-polyoma middle T-antigen (PyMT) murine breast cancer model that depends on c-Src. PyMT mice crossed with Src*/SD mice displayed accelerated tumor formation. The earlier onset of tumors can be explained by the interaction of the Src* domain with PyMT and targeting the fused phosphorylated SD to the membrane. At membrane compartments, it might integrate membrane-associated active signaling complexes leading to increased proliferation measured by phospho-Histone H3 staining. Although these results were unexpected, they emphasize the importance of preventing the membrane association of Src*/SD when employed as decoy molecule.

Hypoxia-response element (HRE)-directed transcriptional regulation of the rat lysyl oxidase gene in response to cobalt and cadmium.

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5'-ACGTG-3') exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10-100 µM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)-treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at -387/-383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.

Transgenic expression of an altered angiotensin type I AT1 receptor resulting in marked modulation of vascular type I collagen.

The angiotensin II (AngII) type I receptor (AT1) was modified by replacing its third intracellular loop and C-terminal tail with the corresponding regions from the bradykinin B2 receptor. Transgenic mice were produced that overexpress this mutated receptor (AB3T). Considerably less collagen content in the intact aorta and in primary aortic smooth muscle cells (aSMCs) cultures was observed in the transgenic mice. On the other hand, elastin content remained unchanged as measured by Western blot, and insoluble amino acid quantitation. The contraction of isolated aortas also remained unaltered. The aSMCs derived from the transgenic mice showed a reduction in AngII responsive type I collagen production. In aSMCs from transgenic mice, the cascade of Akt to the mammalian target rapamycin (mTOR) to p70 S6 kinase (p70S6K) was not AngII activated, while in the aSMCs from wild-type (WT) mice the cascade was AngII activated. Angiotensin activation of Smad2 and Stat3 was also reduced in the AB3T aSMCs. However, no change in the effect of transforming growth factor ß (TGFß) on type I collagen production was observed. Also, the activation of ERK and JNK and G-protein linked signaling remained unaltered in response to AngII. Akt and PI3K activation inhibitors blocked AngII-stimulated type I collagen expression in WT aSMCs, whereas ERK inhibitor had no such effect. Our results point to an Akt/mTOR/p70S6K regulation of collagen production by AngII with participation of Smad2 and Stat3 cascades in this process.

CK2α is essential for embryonic morphogenesis.

CK2 is a highly conserved serine-threonine kinase involved in biological processes such as embryonic development, circadian rhythms, inflammation, and cancer. Biochemical experiments have implicated CK2 in the control of several cellular processes and in the regulation of signal transduction pathways. Our laboratory is interested in characterizing the cellular, signaling, and molecular mechanisms regulated by CK2 during early embryonic development. For this purpose, animal models, including mice deficient in CK2 genes, are indispensable tools. Using CK2α gene-deficient mice, we have recently shown that CK2α is a critical regulator of mid-gestational morphogenetic processes, as CK2α deficiency results in defects in heart, brain, pharyngeal arch, tail bud, limb bud, and somite formation. Morphogenetic processes depend upon the precise coordination of essential cellular processes in which CK2 has been implicated, such as proliferation and survival. Here, we summarize the overall phenotype found in CK2α (-/- ) mice and describe our initial analysis aimed to identify the cellular processes affected in CK2α mutants.

The critical role of the cellular thiol homeostasis in cadmium perturbation of the lung extracellular matrix.

Cadmium (Cd) inhalation can result in emphysema. Cd exposure of rat lung fibroblasts (RFL6) enhanced levels of metal scavenging thiols, e.g., metallothionein (MT) and glutathione (GSH), and the heavy chain of gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, concomitant with downregulation of lysyl oxidase (LO), a copper-dependent enzyme for crosslinking collagen and elastin in the extracellular matrix (ECM). Cd downregulation of LO in treated cells was closely accompanied by suppression of synthesis of collagen, a major structure component of the lung ECM. Using rats intratracheally instilled with cadmium chloride (30 microg, once a week) as an animal model, we further demonstrated that although 2-week Cd instillation induced a non-significant change in the lung LO activity and collagen synthesis, 4- and 6-week Cd instillation resulted in a steady decrease in the lung LO and collagen expression. The lung MT and total GSH levels were both upregulated upon the long-term Cd exposure. Emphysematous lesions were generated in lungs of 6-week Cd-dosed rats. Increases of cellular thiols by transfection of cells with MT-II expression vectors or treatment of cells with GSH monoethyl ester, a GSH delivery system, markedly inhibited LO mRNA levels and catalytic activities in the cell model. Thus, Cd upregulation of cellular thiols may be a critical cellular event facilitating downregulation of LO, a potential mechanism for Cd-induced emphysema.

Loss of AND-34/BCAR3 expression in mice results in rupture of the adult lens.

PURPOSE: AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism. METHODS: AND-34(-/-) mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues. RESULTS: Western analyses confirmed total loss of expression in AND-34(-/-) splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34(-/-) mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34(-/-) mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34(-/-) mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34(+/+) lens epithelial cells, it was markedly reduced in the AND-34(-/-) lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34(+/+) than in AND-34(-/-) lens epithelium. CONCLUSIONS: These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens.

Gene targeting of CK2 catalytic subunits.

Protein kinase CK2 is a highly conserved and ubiquitous serine-threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2beta subunits and two catalytic subunits, either CK2alpha/CK2alpha, CK2alpha/CK2alpha', or CK2alpha'/CK2alpha'. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2alpha' is essential for male germ cell development, and we now demonstrate that CK2alpha has an essential role in embryogenesis, as mice lacking CK2alpha die in mid-embryogenesis, with cardiac and neural tube defects.

Structure and function analysis of the CMS/CIN85 protein family identifies actin-bundling properties and heterotypic-complex formation.

Members of the CMS/CIN85 protein family participate in clathrin-mediated endocytosis and play a crucial role in maintaining the kidney filtration barrier. The CMS protein structure includes three Src homology 3 (SH3) domains and a proline-rich (PR) region that is connected by a 'linker' sequence to a coiled-coil (CC) domain. We show that CMS is a component of special actin-rich adhesion structures--podosomes--and demonstrate specific actin-binding properties of CMS. We have found that the entire C-terminal half of CMS is necessary for efficient binding to filamentous actin (F-actin). CMS and CIN85 can crosslink F-actin into bundles, a function that depends on the PR region and the CC domain. Removal of these domains reduces migration. CMS can also form heterotypic complexes with CIN85. CIN85 is expressed as multiple isoforms that share the CC domain, suggesting that heterotypic interactions with CMS provides a mechanism to regulate CMS binding to F-actin and thus for modulating dynamic rearrangements of the cytoskeleton.

Perturbation of copper (Cu) homeostasis and expression of Cu-binding proteins in cadmium-resistant lung fibroblasts.

To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.

Cloning and characterization of the rat lysyl oxidase gene promoter: identification of core promoter elements and functional nuclear factor I-binding sites.

Lysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5'-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from -78 to -51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5' Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region -709/-598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment -612/-580, which contains a nuclear factor I (NFI)-binding site at the region -594/-580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at -147/-133, were identified in the LO promoter region -804/-1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation.

Angiotensin II type 1 and bradykinin B2 receptors expressed in early stage epithelial cells derived from human embryonic stem cells.

When human embryonic stem (hES) cells were placed into suspension culture followed by culture on BD matrigel coated plates in the presence of medium conditioned by NIH-3T3 cells, they differentiated into cells of which more than 95% stained positive for keratin 8 by day 14, demonstrating that the hES cells had committed to an epithelial lineage. Approximately 50% of the keratin 8 staining cells became positive for cytokeratin 14 after 26 days. Binding experiments supported by real time PCR showed that the expression of bradykinin B2 (BKB2) and angiotensin II type 1 (AT1) receptors accompanied this differentiation. Neither receptor was expressed in the pluripotent H9 stem cells. However, transduction of the hES cells with lentivirus containing BKB2 or AT1R cDNA resulted in ligand binding and ERK1/2 activation but not in Galphai or Galphaq coupled signaling. In the differentiated cells, both BKB2R and AT1R were expressed constitutively and effected typical Galphai and Galphaq coupled signaling characterized by the release of arachidonate, generation of inositol phosphates, and Ca(2+) mobilization. These signals were abolished by the receptor antagonists, losartan, and HOE 140. Angiotensin II and bradykinin also stimulated the phosphorylation of ERK1/2, JNK1/2, and p70S6 in the differentiated cells. Our results demonstrate that human embryonic stem cells can be differentiated effectively into the epithelial lineage and that when differentiated express functional, signaling AT1 and BKB2 receptors.

The A2B adenosine receptor protects against inflammation and excessive vascular adhesion.

Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor-knockout/reporter gene-knock-in (A2BAR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-alpha, and a consequent downregulation of IkappaB-alpha are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.

Neutrophil elastase-initiated EGFR/MEK/ERK signaling counteracts stabilizing effect of autocrine TGF-beta on tropoelastin mRNA in lung fibroblasts.

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938-18946, 2002). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-alpha-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-beta signaling, because TGF-beta type I receptor kinase inhibitor or TGF-beta neutralizing antibody dramatically decreases tropoelastin mRNA and protein levels. Half-life of tropoelastin mRNA in RFL-6 cells is >24 h, but it is decreased to approximately 8 h by addition of TGF-beta neutralizing antibody, EGF, TGF-alpha, or NE. Tropoelastin mRNA destabilization by NE, EGF, or TGF-alpha is abolished by AG1478 or U0126. EGF-dependent tropoelastin mRNA downregulation is reversed upon ligand withdrawal, whereas chronic EGF treatment leads to persistent downregulation of tropoelastin mRNA and protein levels and decreases insoluble elastin deposition. We conclude that NE-initiated EGFR/MEK/ERK signaling cascade overrides the autocrine TGF-beta signaling on tropoelastin mRNA stability and, therefore, decreases the elastogenic response in RFL-6 fibroblasts. We hypothesize that persistent EGFR/MEK/ERK signaling could impede the TGF-beta-induced elastogenesis/elastin repair in the chronically inflamed, elastase/anti-elastase imbalanced lung in emphysema.

Inhibition of the expression of lysyl oxidase and its substrates in cadmium-resistant rat fetal lung fibroblasts.

Copper (Cu)-dependent lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin stabilizing the extracellular matrix (ECM). Chronic inhalation of cadmium (Cd), a toxic metal, induces emphysema. To probe mechanisms of Cd injury to the lung, we developed Cd-resistant (CdR) cells from rat fetal lung fibroblasts (RFL6) by chronic exposure to CdCl(2) from 1 to 40 microM and further examined their expressions of LO, LO substrates, and Cu-scavenging thiols. Levels of cellular thiols, metallothionein, and glutathione in CdR cells were elevated to 13.0- and 3.2-fold of parental controls, respectively, whereas LO mRNA and protein levels were markedly reduced in these cells, with catalytic activity declining to only 16% of the parental control. A conspicuous 52 kDa species rather then the normal 50 kDa proenzyme appeared in the CdR cell extract but not in the conditioned medium, which was codistributed with the endoplasmic reticulum marker [DiOC5(3)] within the cell, implying the Cd-induced 52 kDa species as a product of an abnormal LO-processing defect in secretion. Addition of Cu into CdR cell cultures enhanced the expression of LO mRNA, protein and catalytic activities reflecting limitation of Cu bioavailability for LO in these cells. With inhibition of LO, CdR cells also displayed downregulation of collagen and elastin, substrates of LO. Restoration of collagen synthesis by exposure of CdR cells to purified LO or Cu suggests that inhibition of LO and limitation of Cu cofactor by Cd, as key phenotype changes, accelerated collagen and elastin damage, a critical event pertinent to emphysema pathogenesis.

Transcriptional and posttranscriptional inhibition of lysyl oxidase expression by cigarette smoke condensate in cultured rat fetal lung fibroblasts.

Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0-120 microg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80-120 microg CSC/ml. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 microg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 microg CSC/ml. Moreover, 80-120 microg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema.

Adipocyte accumulation in penile corpus cavernosum of the orchiectomized rabbit: a potential mechanism for veno-occlusive dysfunction in androgen deficiency.

Androgens are deemed to be critical for the development, growth, and maintenance of penile tissue as well as for erectile function. Androgens are also reported to inhibit differentiation of stroma progenitor cells into adipocytes and promote differentiation into smooth muscle. The objective of this study was to investigate whether androgen deprivation results in accumulation of adipocytes in the corpus cavernosum. Mature, New Zealand white male rabbits were subjected to sham surgery (control) or orchiectomy. Two weeks after surgery, erectile function was assessed by monitoring changes in intracavernosal blood pressure (ICP) in response to pelvic nerve stimulation. All ICP measurements were normalized to the mean systemic arterial blood pressure. In parallel studies, penile cross sections from control and orchiectomized rabbits were fixed and stained with either Masson's trichrome or hematoxylin and eosin to assess smooth muscle and connective tissue content. Alternatively, tissue sections were stained with Toluidine blue to assess accumulation of fat-containing cells. Orchiectomy resulted in loss of erectile function and penile atrophy, associated with reduced trabecular smooth muscle and increased connective tissue content. Most strikingly, tissue from orchiectomized animals exhibited accumulation of fat-containing cells (adipocytes) in the subtunical region of the corpus cavernosum. We hypothesize that androgen deprivation promotes differentiation of progenitor stroma cells into an adipogenic lineage producing fat-containing cells, thus altering erectile function.

Downregulation of lysyl oxidase and upregulation of cellular thiols in rat fetal lung fibroblasts treated with cigarette smoke condensate.

Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e., elastin and collagen type I, the major components of cellular thiols, i.e., metallothionein (MT) and glutathione (GSH), and gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, in cigarette smoke condensate (CSC)-treated rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to CSC decreased levels of LO catalytic activity, mRNA, and protein, i.e., the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature enzyme in a dose-dependent manner. In addition, CSC also inhibited the expression of collagen type I and elastin, substrates of LO and important components of the lung ECM. Meanwhile, cellular thiols including MT and GSH as well as gamma-GCS were markedly upregulated in CSC-treated cells. To evaluate modulation of LO expression by cellular thiols, we further examined the effect of increased levels of GSH on LO expression at protein and catalytic levels. Interestingly, exposure of cells to glutathione monoethyl ester, a GSH delivery system, effectively elevated cellular GSH levels and induced a dose-dependent decrease in levels of the protein species and catalytic activity of LO. These results suggest that upregulation by CSC of cellular thiols may play an important role in the downregulation of LO and subsequently destabilization of the lung ECM in CS-induced emphysema.

B-Myb represses elastin gene expression in aortic smooth muscle cells.

B-Myb represses collagen gene transcription in vascular smooth muscle cells (SMCs) in vitro and in vivo. Here we sought to determine whether elastin is similarly repressed by B-Myb. Levels of tropoelastin mRNA and protein were lower in aortas and isolated SMCs of adult transgenic mice expressing the human B-myb gene, driven by the basal cytomegalovirus promoter, compared with age-matched wild type (WT) animals. However, the vessel wall architecture and levels of insoluble elastin revealed no differences. Since elastin deposition occurs early in development, microarray analysis was performed using nontransgenic mice. Aortic levels of tropoelastin mRNA were low during embryonal growth and increased substantially in neonates, whereas B-myb levels varied inversely. Tropoelastin mRNA expression in aortas of 6-day-old neonatal transgenic and WT animals was comparable. Recently, we demonstrated that cyclin A-Cdk2 prevents B-Myb-mediated repression of collagen promoter activity. Cyclin A2 levels were higher in neonatal versus adult WT or transgenic mouse aortas. Ectopic cyclin A expression reversed the ability of B-Myb to repress elastin gene promoter activity in adult SMCs. These results demonstrate for the first time that B-Myb represses SMC elastin gene expression and that cyclin A plays a role in the developmental regulation of elastin gene expression in the aorta. Furthermore, the findings provide additional insight into the mechanism of B-myb-mediated resistance to femoral artery injury.

A(3) adenosine receptor deficiency does not influence atherogenesis.

Atherosclerosis is a multifactorial disease, the progression of which is modulated by several factors, including inflammation and hypercholesterolemia. The A(3) adenosine receptor (A(3)AR) has been reported to affect mast cell degranulation leading to inflammation, as well as to influence cardiovascular homeostasis. Here, we show that its deletion can also impact vascular smooth muscle cell (VSMC) proliferation in vitro. Based on these observations, we hypothesized that A(3)AR deficiency would affect atheromatous lesion development in vivo. Our results indicate that the expression of the matrix enzyme lysyl oxidase (LO) is increased while the proliferation potential of VSMC is decreased in A(3)AR-null aortas. This is in accordance with the previously reported inverse correlation between LO level and proliferation. Nevertheless, we found that A(3)-deficiency does not protect vessels against atherogenesis. This was demonstrated in mouse models of high fat diet-induced atherosclerosis and guidewire-induced femoral artery injury. We conclude that the contributions of the A(3)AR to inflammation and to modulating LO levels are not significant enough to control vascular response to injury.