After Bone Marrow Transplantation, the Cell-Intrinsic Th2 Pathway Promotes Recipient T Lymphocyte Survival and Regulates Graft-versus-Host Disease.

Recipient T cells can aggravate or regulate lethal and devastating graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In this context, we have shown before that intestinal immune conditioning with helminths is associated with survival of recipient T cells and Th2 pathway-dependent regulation of GVHD. We investigated the mechanism of survival of recipient T cells and their contribution to GVHD pathogenesis in this helminth infection and BMT model after myeloablative preparation with total body irradiation in mice. Our results indicate that the helminth-induced Th2 pathway directly promotes the survival of recipient T cells after total body irradiation. Th2 cells also directly stimulate recipient T cells to produce TGF-ß, which is required to regulate donor T cell-mediated immune attack of GVHD and can thereby contribute to recipient T cell survival after BMT. Moreover, we show that recipient T cells, conditioned to produce Th2 cytokines and TGF-ß after helminth infection, are fundamentally necessary for GVHD regulation. Taken together, reprogrammed or immune-conditioned recipient T cells after helminth infection are crucial elements of Th2- and TGF-ß-dependent regulation of GVHD after BMT, and their survival is dependent on cell-intrinsic Th2 signaling.

Helminth-Induced Production of TGF-ß and Suppression of Graft-versus-Host Disease Is Dependent on IL-4 Production by Host Cells.

Helminths stimulate the secretion of Th2 cytokines, like IL-4, and suppress lethal graft-versus-host disease (GVHD) after bone marrow transplantation. This suppression depends on the production of immune-modulatory TGF-ß and is associated with TGF-ß-dependent in vivo expansion of Foxp3+ regulatory T cells (Treg). In vivo expansion of Tregs is under investigation for its potential as a therapy for GVHD. Nonetheless, the mechanism of induced and TGF-ß-dependent in vivo expansion of Tregs, in a Th2 polarized environment after helminth infection, is unknown. In this study, we show that helminth-induced IL-4 production by host cells is critical to the induction and maintenance of TGF-ß secretion, TGF-ß-dependent expansion of Foxp3+ Tregs, and the suppression of GVHD. In mice with GVHD, the expanding donor Tregs express the Th2-driving transcription factor, GATA3, which is required for helminth-induced production of IL-4 and TGF-ß. In contrast, TGF-ß is not necessary for GATA3 expression by Foxp3+ Tregs or by Foxp3- CD4 T cells. Various cell types of innate or adaptive immune compartments produce high quantities of IL-4 after helminth infection. As a result, IL-4-mediated suppression of GVHD does not require invariant NKT cells of the host, a cell type known to produce IL-4 and suppress GVHD in other models. Thus, TGF-ß generation, in a manner dependent on IL-4 secretion by host cells and GATA3 expression, constitutes a critical effector arm of helminthic immune modulation that promotes the in vivo expansion of Tregs and suppresses GVHD.

Recirculating Immunocompetent Cells in Colitic Mice Intensify Their Lung Response to Bacterial Endotoxin.

BACKGROUND: Patients with inflammatory bowel disease have higher incidence of airway hyperresponsiveness compared to the general population. Lung inflammation leading to airway hyperresponsiveness causes illnesses for more than ten percent of the population in USA. AIMS: We investigated the lung response to bacterial endotoxin in colitic mice. METHODS: Rag-1 mice were transplanted with negatively selected splenic T cells. Some mice groups were treated with NSAID to develop colitis. All mice were treated with bacterial endotoxin and necropsied 3 weeks later. RESULTS: Colitic mice developed intensified lung inflammation on day 21 of treatment with bacterial endotoxin. Pulmonary lymphocytes from colitic mice displayed a proinflammatory cytokine profile, expressed high ICAM1 and low FoxP3. CD11c+, CD8+ cells bound and responded to non-systemic antigens from gut-localized microbiota and had higher expression of TLR4. CONCLUSIONS: Colitic mice developed exacerbated lung inflammation in response to bacterial endotoxin compared to non-colitic mice. Proinflammatory cytokines from pulmonary lymphocytes induced high expression of ICAM1 and suppressed FoxP3 on CD4+ cells. CD11c+, CD8+ cells binding and responding to gut-localized antigens as well as high expression of TLR4 indicate innate and adaptive lung response to bacterial endotoxin. Inflammatory cells from colons of colitic mice homed in the lungs as well as the intestine suggesting recirculation of sensitized immunocompetent cells. These data support our hypothesis that colitis intensifies lung inflammation.

Sirtuin 1 regulates cardiac electrical activity by deacetylating the cardiac sodium channel.

The voltage-gated cardiac Na+ channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na+ current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.

Oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) regulates vascular smooth muscle migration and apoptosis.

Activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and reactive oxygen species (ROS) promote neointimal hyperplasia after vascular injury. CaMKII can be directly activated by ROS through oxidation. In this study, we determined whether abolishing the oxidative activation site of CaMKII alters vascular smooth muscle cell (VCMC) proliferation, migration and apoptosis in vitro and neointimal formation in vivo. VSMC isolated from a knock-in mouse with oxidation-resistant CaMKIIδ (CaMKII M2V) displayed similar proliferation but decreased migration and apoptosis. Surprisingly, ROS production and expression of the NADPH oxidase subunits p47 and p22 were decreased in M2V VSMC, whereas superoxide dismutase 2 protein expression was upregulated. In vivo, after carotid artery ligation, no differences in neointimal size or remodeling were observed. In contrast to VSMC, CaMKII expression and autonomous activity were significantly higher in M2V compared to WT carotid arteries, suggesting that an autoregulatory mechanism determines CaMKII activity in vivo. Our findings demonstrate that preventing oxidative activation of CaMKII decreases migration and apoptosis in vitro and suggest that CaMKII regulates ROS production. Our study presents novel evidence that CaMKII expression in vivo is regulated by a negative feedback loop following oxidative activation.

Oxidized Ca(2+)/calmodulin-dependent protein kinase II triggers atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is a growing public health problem without adequate therapies. Angiotensin II and reactive oxygen species are validated risk factors for AF in patients, but the molecular pathways connecting reactive oxygen species and AF are unknown. The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a reactive oxygen species-activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ could contribute to AF. METHODS AND RESULTS: We found that oxidized CaMKII was increased in atria from AF patients compared with patients in sinus rhythm and from mice infused with angiotensin II compared with mice infused with saline. Angiotensin II-treated mice had increased susceptibility to AF compared with saline-treated wild-type mice, establishing angiotensin II as a risk factor for AF in mice. Knock-in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A, an enzyme that reduces oxidized CaMKII, were resistant to AF induction after angiotensin II infusion. CONCLUSIONS: Our studies suggest that CaMKII is a molecular signal that couples increased reactive oxygen species with AF and that therapeutic strategies to decrease oxidized CaMKII may prevent or reduce AF.

The multifunctional Ca²âº/calmodulin-dependent kinase IIδ (CaMKIIδ) regulates arteriogenesis in a mouse model of flow-mediated remodeling.

OBJECTIVE: Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca²âº/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis. METHODS AND RESULTS: Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ-/-) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ-/- mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ-/- mice normalized flow-mediated remodeling. CONCLUSION: CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.

Diabetes increases mortality after myocardial infarction by oxidizing CaMKII.

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca(2+)/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.

Oxidation of CaMKII determines the cardiotoxic effects of aldosterone.

Excessive activation of the ß-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II-mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.

I(f) and SR Ca(2+) release both contribute to pacemaker activity in canine sinoatrial node cells.

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a 'voltage clock' and a Ca(2+) dependent process, or 'Ca(2+) clock.' The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (I(f)) is thought to be particularly important. A Ca(2+) dependent process triggers APs when sarcoplasmic reticulum (SR) Ca(2+) release activates inward current carried by the forward mode of the electrogenic Na(+)/Ca(2+) exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca(2+) clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective I(f) antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest ( approximately 14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca(2+) release, but did not affect basal or isoproterenol-enhanced I(f). Taken together, these results indicate that voltage and Ca(2+) dependent automaticity mechanisms coexist in canine SAN cells, and suggest that I(f) and SR Ca(2+) release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.

Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart.

Myocardial Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-kappaB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-kappaB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb-/- mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes.

Calmodulin kinase II is required for fight or flight sinoatrial node physiology.

The best understood "fight or flight" mechanism for increasing heart rate (HR) involves activation of a cyclic nucleotide-gated ion channel (HCN4) by beta-adrenergic receptor (betaAR) agonist stimulation. HCN4 conducts an inward "pacemaker" current (I(f)) that increases the sinoatrial nodal (SAN) cell membrane diastolic depolarization rate (DDR), leading to faster SAN action potential generation. Surprisingly, HCN4 knockout mice were recently shown to retain physiological HR increases with isoproterenol (ISO), suggesting that other I(f)-independent pathways are critical to SAN fight or flight responses. The multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) is a downstream signal in the betaAR pathway that activates Ca(2+) homeostatic proteins in ventricular myocardium. Mice with genetic, myocardial and SAN cell CaMKII inhibition have significantly slower HRs than controls during stress, leading us to hypothesize that CaMKII actions on SAN Ca(2+) homeostasis are critical for betaAR agonist responses in SAN. Here we show that CaMKII mediates ISO HR increases by targeting SAN cell Ca(2+) homeostasis. CaMKII inhibition prevents ISO effects on SAN Ca(2+) uptake and release from intracellular sarcoplasmic reticulum (SR) stores that are necessary for increasing DDR. CaMKII inhibition has no effect on the ISO response in SAN cells when SR Ca(2+) release is disabled and CaMKII inhibition is only effective at slowing HRs during betaAR stimulation. These studies show the tightly coupled, but previously unanticipated, relationship of CaMKII to the betaAR pathway in fight or flight physiology and establish CaMKII as a critical signaling molecule for physiological HR responses to catecholamines.

A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation.

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5.

Embryonic organs attain their final dimensions through the generation of proper cell number and size, but the control mechanisms remain obscure. Here, we establish Gridlock (Grl), a Hairy-related basic helix-loop-helix (bHLH) transcription factor, as a negative regulator of cardiomyocyte proliferative growth in zebrafish embryos. Mutations in grl cause an increase in expression of a group of immediate-early growth genes, myocardial genes, and development of hyperplastic hearts. Conversely, cardiomyocytes with augmented Grl activity have diminished cell volume and fail to divide, resulting in a marked reduction in heart size. Both bHLH domain and carboxyl region are required for Grl negative control of myocardial proliferative growth. These Grl-induced cardiac effects are counterbalanced by the transcriptional activator Gata5 but not Gata4, which promotes cardiomyocyte expansion in the embryo. Biochemical analyses show that Grl forms a complex with Gata5 through the carboxyl region and can repress Gata5-mediated transcription via the bHLH domain. Hence, our studies suggest that Grl regulates embryonic heart growth via opposing Gata5, at least in part through their protein interactions in modulating gene expression.

Death, cardiac dysfunction, and arrhythmias are increased by calmodulin kinase II in calcineurin cardiomyopathy.

BACKGROUND: Activation of cellular Ca2+ signaling molecules appears to be a fundamental step in the progression of cardiomyopathy and arrhythmias. Myocardial overexpression of the constitutively active Ca2+-dependent phosphatase calcineurin (CAN) causes severe cardiomyopathy marked by left ventricular (LV) dysfunction, arrhythmias, and increased mortality rate, but CAN antagonist drugs primarily reduce hypertrophy without improving LV function or risk of death. METHODS AND RESULTS: We found that activity and expression of a second Ca2+-activated signaling molecule, calmodulin kinase II (CaMKII), were increased in hearts from CAN transgenic mice and that CaMKII-inhibitory drugs improved LV function and suppressed arrhythmias. We devised a genetic approach to "clamp" CaMKII activity in CAN mice to control levels by interbreeding CAN transgenic mice with mice expressing a specific CaMKII inhibitor in cardiomyocytes. We developed transgenic control mice by interbreeding CAN transgenic mice with mice expressing an inactive version of the CaMKII-inhibitory peptide. CAN mice with CaMKII inhibition had reduced risk of death and increased LV and ventricular myocyte function and were less susceptible to arrhythmias. CaMKII inhibition did not reduce transgenic overexpression of CAN or expression of endogenous CaMKII protein or significantly reduce most measures of cardiac hypertrophy. CONCLUSIONS: CaMKII is a downstream signal in CAN cardiomyopathy, and increased CaMKII activity contributes to cardiac dysfunction, arrhythmia susceptibility, and longevity during CAN overexpression.

[Preparation and functional study of an adenovirus vector expressing human plasminogen kringle 5 gene].

Tumor angiogenesis plays a pivotal role in the progress of tumor. Among the various endogenous angiogenic inhibitors discovered, the human plasminogen kringle 5 (K5) has been demonstrated to be a potential inhibitor of the proliferation and migration of vascular endothelial cells in vitro. The replication-incompetent adenovirus (Ad) vector Adeno-X-CMV-K5 (Ad-K5) (where CMV is cytomegalovirus) was constructed and its antiangiogenic effect was tested on vascular endothelial cell and tumor cell. For the construction, the K5 cDNA was fused in-frame with human plasminogen signal sequence and inserted into the eukaryotic expression vector pcDNA3 to form pcDNA3K5. The recombinant plasmid was subcloned into the shuttle plasmid pShuttle under the control of the constitutive CMV immediate-early promoter. The plasmid carrying the cDNA for K5 (pShuttleKS) was then recombined with the Adeno-X viral DNA and transformed into E. coli DH5alpha. The resultant recombinant plasmid pAd-K5 was transfected into human embryonic kidney (HEK) 293 cells with liposome. The adenovirus expressing human plasminogen kringle 5 (Ad-K5) was successfully packaged and propagated in 293 cells, as detected by the cytopathic effect (CPE) on the cells, and the viral titer in the supernatant was 5 x 10(8) pfu/mL by plaque assay. Both human umbilical vein endothelial cell line ECV304 and human breast carcinoma cell line MDA-MB-231 were infected with Ad-K5 and Ad-LacZ, which was used the negative control, and assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared with uninfected control and Ad-LacZ infected control, Ad-K5 infected group at 80 MOI (multiplicity of infection) significantly inhibited ECV304 proliferation; the difference between uninfected control and Ad-LacZ infected control was not significant. In contrast, there was no significant difference in the proliferation of MDA-MB-231 among all the treatments. In addition, the Ad-K5 at 100 MOI inhibited the differentiation and tube formation of ECV304 on ECMatrix gel. These results suggested that the recombinant replication-defective Adenovirus expressing human plasminogen kringle 5 inhibited the proliferation, differentiation and tube formation of ECV304 and had no effect on the proliferation of MDA-MB-231. Adenovirus mediated human plasminogen kringle 5 gene therapy may be a potential treatment of cancer through angiogenesis inhibition.

[Preliminary study of ALK3 downstream genes related to ventricular septum defect].

To investigate the function of ALK3 gene, the gene regulation and the signaling pathway related to ventricular septum defect during heart development. The model mice with ALK3 gene knock-out via alpha-MHC-Cre/lox P system were bred. The mRNA expression level of control group was compared with that of experiment group and ALK3 downstream genes were screened using PCR-select cDNA subtraction microarray. The mRNA of control group was extracted from E11.5 normal mouse hearts, and that of experiment group, from E11.5 hearts of mice with alpha-MHC Cre(+/-) ALK3(F/+) genotype. It was found that the mice with ALK3 gene knock-out produced heart defects involving the interventricular septum. The platelet-activating factors acetylhydrolase and the transcription factor Pax-8 and so on, were down-regulated. However, the Protein Tyrosine Kinase (PTK) of Focal Adhesion Kinase (FAK) subfamily and beta subtype protein 14-3-3 were up-regulated in the alpha-MHC Cre(+/-) ALK3(F/-) mice. These data provide support that ALK3 gene played an important role during heart development. The platelet-activating factors acetylhydrolase and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and beta subtype protein 14-3-3 might be regulatory factors in this pathway.

[A study on myocardial Pax-8 gene].

OBJECTIVE: Conventional deletion of ALK3, also termed as bone morphogenetic protein (BMP) receptor IA, in mice might result in early embryonic lethality. To investigate the function of ALK3 in cardiac development, the cardiac-specific deletion of ALK3 in mice was made by Dr. Schneider, using Cre recombinase driven by the alpha-MHC promoter that Dr. Fukushipe worked out. Such specific deletion of ALK3 caused death in mid-gestation with defects in the trabeculae, interventricular septum, and endocardial cushion. Since ALK3 is not a cardiac-specific gene, it is extremely important to identify ALK3 downstream genes. METHODS: Alpha-MHC Cre+/-, ALK3 F/- and alpha-MHC Cre+/-, ALK3 F/+ embryos were obtained after 20 alpha-MHC Cre+/-, ALK3 +/- mice and 20 ALK3 F/F mice were mating. The ALK3 downstream genes were screened using microarray made in Germany that could identify 25000 genes in mouse. Two populations of mRNA, one derived from the embryonic heart (11.5 days) of alpha-MHC Cre+/-, ALK3 F/- mice, and the other derived from the alpha-MHC Cre+/-, ALK3 F/+ mice, were compared. Cardiac-specific ALK3 downstream genes were identified using real time quantitative RT-PCR and in situ hybridization. RESULTS: The expression of 12 genes, such as Pax-8 and Hox-3.5 were down-regulated in alpha-MHC Cre+/-, ALK3 F/- mouse heart. The expression of 16 genes including Ras-related protein Rab-5b and EPS-8 protein was up-regulated in the group of alpha-MHC Cre+/-, ALK3 F/-. It was found that the Box protein Pax-8 gene was down-regulated by 7.1 fold (P < 0.001) in the alpha-MHC Cre+/-, ALK3 F/- mice by real time quantitative RT-PCR. It was also revealed that the Box protein Pax-8 gene was expressed stronger in alpha-MHC Cre+/-, ALK3 F/+ than alpha-MHC Cre+/-, ALK3 F/- E11.5 days mouse heart by means of in situ hybridization. CONCLUSION: The Box protein Pax-8 gene is an important and cardiac-specific ALK3 downstream gene in the BMP signaling pathway during inter-ventricular septum development.

Recombinant human heparin-binding neurite-promoting factor expressed with yeast stimulates neurites outgrowth.

OBJECTIVES: Heparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro. We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro. METHODS: cDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method. After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system. The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening. Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation. His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free. His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418. PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome. Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth. RESULTS: In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data. The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K. SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature. The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells. CONCLUSION: Recombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.