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J. Biol. Chem., Vol. 277, Issue 25, 22734-22742, June 21, 2002

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Inositol Polyphosphate 1-Phosphatase Is a Novel Antihypertrophic Factor^*

Elizabeth A. Woodcock, Bing Hui Wang, Jane F. Arthur, Alicia Lennard, Scot J. Matkovich^§, Xiao-Jun Du^¶, Joan Heller Brown, and Ross D. Hannan^**

From the Cellular Biochemistry Laboratory, ^¶ Experimental Cardiology Laboratory, and ^** Gene Transcription Laboratory, Baker Medical Research Institute, PO Box 6492, St. Kilda Road Central, Melbourne, 8008, Victoria, Australia and the  Department of Pharmacology, University of California, San Diego, La Jolla, California 92037-0636

Received for publication, October 30, 2001, and in revised form, March 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of G_q-coupled ₁-adrenergic receptors leads to hypertrophic growth of neonatal rat ventricular cardiomyocytes that is associated with increased expression of hypertrophy-related genes, including atrial natriuretic peptide (ANP) and myosin light chain-2 (MLC), as well as increased ribosome synthesis. The role of inositol phosphates in signaling pathways involved in these changes in gene expression was examined by overexpressing inositol phosphate-metabolizing enzymes and determining effects on ANP, MLC, and 45 S ribosomal gene expression following co-transfection of appropriate reporter gene constructs. Overexpression of enzymes that metabolize inositol 1,4,5-trisphosphate did not reduce ANP or MLC responses, but overexpression of the enzyme primarily responsible for metabolism of inositol 4,5-bisphosphate (Ins(1,4)P₂), inositol polyphosphate 1-phosphatase (INPP), reduced ANP and MLC responses associated with ₁-adrenergic receptor-mediated hypertrophy. Similarly overexpressed INPP reduced ANP and MLC responses associated with contraction-induced hypertrophy. In addition, overexpression of INPP reduced the increase in ribosomal DNA transcription associated with both hypertrophic models. Hypertrophied cells from both cell models as well as ventricular tissue from mouse hearts hypertrophied by pressure overload in vivo contained heightened levels of Ins(1,4)P₂, suggesting reduced INPP activity in three different models of hypertrophy. These studies provide evidence for an involvement of Ins(1,4)P₂ in hypertrophic signaling pathways in ventricular myocytes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cardiac myocytes respond to stressors or to growth stimuli by increasing cell size in the absence of substantial cell division (1). Such hypertrophic growth in the in vivo situation is initially advantageous in allowing the heart to compensate for such factors as loss of myocytes or increased aortic pressure. However, sustained hypertrophy eventually leads to the development of heart failure, currently a leading cause of death in western societies (2). For this reason, the mechanisms involved in regulating hypertrophic growth are the subject of intense investigation.

Signaling pathways involved in cardiac hypertrophic growth appear to be similar to those that mediate growth and division in other cell types (3). Hypertrophy can be initiated by cytokines, growth factors, or factors that activate G protein-coupled receptors, principally those that activate the G_q class of heterotrimeric G proteins (1). Activation of G_q either following receptor activation or directly by the use of constitutively active G_q mutants leads to hypertrophic responses involving mitogen-activated protein kinase pathways as well as monomeric G proteins, although the details of all the intermediates are not fully understood (4). Currently the only well established substrates for activated G_q are the isoforms of PLC-¹ (5), thus activation of G_q would be expected to lead to sn-1,2-diacylglycerol generation and protein kinase C (PKC) activation as well as Ins(1,4,5)P₃ production and increases in cytosolic Ca²⁺. While activation of PKC can initiate hypertrophic responses, PKC does not appear to be solely responsible for all aspects of G_q-initiated hypertrophic responses (6, 7), implying a role for the inositol phosphate (InsP) arm of the pathway. Recent studies have identified mechanisms whereby raised cytosolic Ca²⁺ could lead to hypertrophic responses via the Ca²⁺-dependent phosphatase calcineurin or possibly via pathways initiated by calmodulin-activated kinase IV (8-10). However, Ins(1,4,5)P₃ has little effect on global Ca²⁺ levels in myocardial preparations, and such effects would need to be considered against the large increases and decreases in cytosolic Ca²⁺ that occur during each beat in contracting myocytes. There is no evidence that Ins(1,4,5)P₃ can cause sustained increases in diastolic Ca²⁺ levels as would be required for calcineurin or calmodulin-activated kinase mechanisms (11, 12).

We have previously presented evidence that activation of G_q-coupled receptors in cardiomyocytes leads to an InsP response that involves principally the generation of Ins(1,4)P₂ from PtdIns(4)P independently of any Ins(1,4,5)P₃ production (13, 14). Such a mechanism might serve to reduce the generation of Ins(1,4,5)P₃ because that is potentially arrhythmogenic (12, 15-17). However, it is also possible that products of these Ins(1,4)P₂-generating pathways have a functional significance of their own. In the current paper we present evidence that inositol polyphosphate 1-phosphatase (INPP), the enzyme primarily responsible for metabolism of Ins(1,4)P₂, reduces hypertrophic responses in neonatal rat cardiomyocytes. This is the first report of a functional importance of Ins(1,4)P₂ in cardiac signaling pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Isolation and Culture-- Neonatal cardiomyocytes (NCMs) were isolated from 1-3-day-old rats and maintained in serum-free Dulbecco's modified Eagle's medium supplemented with 10 µg/ml insulin and 10 µg/ml transferrin exactly as described previously (18). Bromodeoxyuridine (BrdUrd, 0.1 mM) was included for the first 3 days.

Expression Constructs and Cloning of Human INPP-- INPP was cloned from a human heart cDNA library (Stratagene) by PCR using primers corresponding to positions 1-22 and 1177-2000, sense and antisense, respectively, of the published human INPP cDNA sequence (19). The 5'-primer contained an additional 24 nucleotides coding for the FLAG epitope (Sigma) inserted in-frame between the start ATG and the second codon. The PCR product was ligated into the pCR3.1 mammalian expression vector (Invitrogen) resulting in the construct pCR3.1-FLAG-INPP that drives expression of FLAG-tagged INPP (sense orientation) under control of the cytomegalovirus (CMV) promoter. Plasmids containing full-length antisense inserts were also selected. Orientation of the INPP insert was determined by sequencing and endonuclease mapping.

The atrial natriuretic peptide (ANP)-luciferase plasmid (pANP(638)L5'), the myosin light chain-2 (MLC)-luciferase plasmid (pMLC(250)L5'), the AP-1-luciferase plasmid (TRE2PRL(36)), the CMV--galactosidase, and the reporter construct for ribosomal gene transcription, pSMECAT, have been described previously (20, 21). The pCR3.1 vector containing the chloramphenicol acetyltransferase (CAT) gene or the -galactosidase gene was used as control in experiments involving INPP. The expression plasmid encoding the type 1 Ins(1,4,5)P₃ 5-phosphatase (22) was obtained from Prof. Christina Mitchell (Monash University). The plasmid encoding the A isoform of Ins(1,4,5)P₃ 3-kinase was obtained from Dr. Christophe Erneux (Free University of Brussels, Belgium). Plasmids encoding constitutively active JNK-1 and c-Jun were obtained from Dr. N. Dhanasekaran (Temple University School of Medicine, Philadelphia, PA), and the plasmid expressing the AT-1_A receptor was supplied by Dr. Walter Thomas (Baker Institute).

SDS-PAGE and Western Blotting-- Samples containing 30 µg of protein were separated electrophoretically on 10% SDS-PAGE gels under reducing conditions and were subsequently transferred to nitrocellulose membranes. Membranes were incubated with monoclonal anti-FLAG antibody (M2, Sigma) at a dilution of 1 in 2000 or polyclonal anti-rabbit antiserum against INPP at a dilution of 1 in 5000 followed by incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Bio-Rad). Detection was achieved using the enhanced chemiluminescence method (Amersham Biosciences) according to the manufacturer's instructions. The molecular sizes of the immunodetected proteins were verified by comparison to the migration of prestained protein markers (Bio-Rad) electrophoresed in parallel.

Antibody Preparation-- The entire coding region of the INPP cDNA was amplified using primers that introduced a His₆ leader after the ATG and then cloned into the inducible bacterial expression vector pET17b (Novagen). Positive clones were verified by sequencing. Recombinant protein was induced with isopropyl-1-thio--D-galactopyranoside and purified using Ni²⁺ affinity resins as recommended by the supplier (Qiagen). The purified protein was used to generate antisera in rabbits, and the sera were purified as described elsewhere (23).

Verification of INPP Expression and Activity-- Expression of recombinant INPP was confirmed by Western analysis and enzyme assays on transfected cells. CHO cells transfected with pCR3.1-FLAG-INPP (LipofectAMINE, Invitrogen) expressed a protein of molecular mass 47 kDa recognized by anti-FLAG antibody that was not observed in cells transfected with pCR3.1-CAT, a construct that expresses the non-mammalian protein chloramphenicol acetyltransferase (Fig. 1A). The identity of the immunoreactive protein as INPP was further verified by blotting with affinity purified anti-INPP antibody that also identified an immunoreactive protein of a size consistent with the molecular mass predicted from the amino acid sequence of INPP. The expression of this protein was increased by transfection with pCR3.1-FLAG-INPP. Transfection of CHO cells with pCR3.1-FLAG-INPP also increased INPP activity, which was measured as previously described (24) (Fig. 1A). To ensure that overexpression of INPP perturbed InsP metabolism, we transfected HEK-293 cells with Rc/CMV-AT-1_A to overexpress AT-1_A receptors as well as with pCR3.1-FLAG-INPP or vector control. Cells were subsequently labeled with [³H]inositol (20 µCi/ml) for 24 h and then stimulated for 20 min with angiotensin II (1 µM) in the presence of LiCl (10 mM). [³H]InsPs were extracted and quantified by HPLC as described below. Overexpression of INPP caused a selective decrease in Ins(1,4)P₂ (Fig. 1B). Thus, transfection with pCR3.1-FLAG-INPP causes increased expression of active INPP that leads to reduced Ins(1,4)P₂ content.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Overexpression of INPP increases protein expression and enzyme activity and decreases Ins(1,4)P2 content. A, CHO cells were transfected with pCR3.1-FLAG-INPP or vector as described under "Experimental Procedures," and cell-free extracts were prepared. Upper panels, extracts were subjected to SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies to FLAG or to recombinant INPP as indicated. Molecular weight markers are shown. All experiments were performed at least three times with similar results. Lower panel, extracts were assayed for INPP activity, which is expressed as nmol of Ins(1,4)P2 hydrolyzed/min/mg of protein. Values shown are mean ± S.E. of triplicate estimations. *, p < 0.01 relative to control vector. B, HEK-293 cells overexpressing AT-1A angiotensin receptors were transfected with pCR3.1-FLAG-INPP or control vector, labeled with [3H]inositol, and stimulated with angiotensin II for 20 min. [3H]InsPs were extracted and quantified. Peaks corresponding to Ins(1,4)P2 are indicated. INPP overexpression reduced [3H]Ins(1,4)P2 levels from 1714 ± 285 to 797 ± 69 (mean ± S.E., n = 3, p < 0.01).

Hypertrophic Models-- For phenylephrine (PE)-induced hypertrophy, NCMs were plated at 400 cells/mm² on 35-mm wells and treated with 50 µM PE and 1 µM propranolol for 40 h. Control cells were treated with propranolol only. Under these conditions cardiomyocytes do not spontaneously contract. For contraction-induced hypertrophy, cardiomyocytes where plated at 1600 cells/mm². Under these conditions cardiomyocytes spontaneously contract and undergo hypertrophic growth compared with non-contracting cells (25-27). Control cells were contraction-arrested by the addition of 50 mM KCl to the medium to depolarize the cells. Hypertrophy in contraction-arrested cells was initiated by reducing the level of KCl in the medium from 50 to 5 mM. Spontaneous contraction occurred within 1 h of the reduction in KCl. Protein content of control and hypertrophied cells from both models are shown in Table I.

For producing pressure overload hypertrophy, mice (F1 crosses of C57/SJL strains) were anesthetized by intraperitoneal injection of a mixture of ketamine (8 mg/100 g), xylazine (2 mg/100 g), atropine (0.6 mg/100 g), and carprofen (0.5 mg/100 g). The chest was opened along the midline of the upper sternum. The segment of aortic arch between the right innominate and the left main carotid arteries was dissected, and the aortic diameter was narrowed by 70% (28). Sham-operated animals underwent a similar operation except that the aortic arch was not constricted. All experiments were carried out 6 weeks after surgery when hypertrophy was well established (28) (Table I).

Transient Transfection and Reporter Gene Activity-- Transfection experiments were performed in triplicate using cardiomyocytes on 35-mm wells 1 day after isolation. Transient transfection using a total of 4.8 µg of DNA/well was performed by the calcium phosphate method. As described elsewhere, this results in transfection efficiency between 1 and 2% (29). Cells were harvested into Lysis Buffer containing 0.1 M K₂HPO₄, 1% Triton X-100, 1 mM dithiothreitol, pH 7.9 (luciferase assays) or 0.25 M Tris-Cl, pH 8.0 (CAT assays). Luciferase activity was measured in 100 mM Tricine, 10 mM MgSO₄, 2 mM EDTA, 2 mM ATP, 75 µM luciferin, pH 7.8 for 2 min in a Lumat LB9507 Luminometer. CAT activity in cell extracts was measured by the synthesis of acetylated [¹⁴C]chloramphenicol, analyzed by TLC as described previously (30), and quantified using a Fujix BAS 1000 phosphorimaging system. -Galactosidase activity was measured by using o-nitrophenyl--D-galactoside (0.25 mM) as substrate and determining absorbance at 410 nm. Protein concentration was assayed using the Bradford method. To control for any differences in transfection efficiency, data were expressed as luciferase activity relative to the activity of -galactosidase co-expressed under a CMV promoter.

[³H]InsP Responses in NCMs and in Mouse Left Ventricle-- NCMs on 35-mm wells were labeled with [³H]inositol (15 µCi/ml) in inositol-free Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, and BrdUrd for 48 h. Cells were then washed with non-radioactive medium and incubated in Dulbecco's modified Eagle's medium containing 1 µM propranolol and 10 mM LiCl for 10 min prior to addition of 100 µM norepinephrine (NE) for 20 min. [³H]InsPs were extracted using 5% trichloroacetic acid, 5 mM phytic acid, 2.5 mM EDTA and centrifuged, and the supernatant was extracted with 0.75 volumes of trichloroethane:tri-N-octylamine (1:1, v/v) as described previously (31). Aqueous phases were treated with proteinase K (2.5 µg/ml), passed through Dowex-50 columns, and lyophilized. [³H]InsPs were separated using anion exchange HPLC and quantified using an on-line -counter as described previously (32).

Mouse left ventricle strips (15-20 mg of tissue) were mounted in 3-ml organ baths in Hepes-buffered Krebs' medium and labeled with [³H]inositol (20 µCi/ml) as described previously (28). Labeled strips were stimulated with 100 µM NE in the presence of propranolol and LiCl for 20 min followed by rapid freezing in liquid N₂. [³H]InsPs were extracted and quantified as described above.

Treatment of Data-- Differences between treatment groups were assessed by one-way analysis of variance with Tukey's test for multiple comparisons and accepted as statistically significant at a family error rate of p < 0.05 (individual pairwise comparisons were significant at p < 0.02). Unless otherwise noted, results shown are from representative experiments performed in triplicate that were repeated in independent NCM preparations at least three times.

Materials-- Fetal calf serum specially selected for low endotoxin was obtained from the Commonwealth Serum Laboratories, Parkville, Australia. Dulbecco's modified Eagle's medium, Hepes, and other materials for the preparation of cell culture solutions and media were cell culture grade, obtained from Sigma, and dissolved in milliQ H₂O. Norepinephrine bitartrate and phenylephrine were purchased from Sigma. [³H]Inositol (18.00 Ci/mmol) was obtained from Amersham Biosciences. Other reagents were obtained from Sigma or BDH/AnalaR and were of analytical reagent grade.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PE-stimulated ANP Responses Require G_q and PLC-- NCMs were transfected with expression plasmids encoding G_q wild type (WT) or G_q mutants together with the reporter gene ANP-luciferase as a measure of hypertrophic signaling and CMV--galactosidase to control for transfection efficiency. 20 h after transfection, PE (50 µM) was added for a further 40 h. Cells were harvested, and luciferase and -galactosidase activities were measured. PE caused a substantial increase in ANP expression as indicated by luciferase activity in transfected cells (Fig. 2). As reported previously, the PE response was increased by overexpression of G_q-WT, while basal activity was not altered (29). Expression of the constitutively active mutant G_q(Q209L) increased ANP expression, and there was no further increase with added PE. In marked contrast, G_q(Q209L) with alanine substitutions D243A, N244E, and E245A (QL,DNE) rendering it unable to stimulate PLC (33) had no detectable effect on ANP responses in the presence or absence of PE. Thus both G_q and PLC appear to be involved in mediating ANP responses to PE in cardiomyocytes.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Stimulation of ANP promoter activity by PE involves Gq and PLC activation. NCMs were transfected with Gq-WT, constitutively active Gq (Gq(Q209L)), constitutively active Gq that is unable to activate PLC (Gq(QL,DNE)), or control vector and subsequently stimulated with 50 µM PE. Luciferase and -galactosidase activities were measured after 40 h. Values shown are luminometer readings per unit of -galactosidase activity expressed as absorbance at 410 nm (mean ± S.E.) of triplicate estimations. The experiment was performed three times with similar results. Open bars, no additions; black bars, PE. *, p < 0.01 relative to no additions. , p < 0.01 relative to PE stimulation in cells transfected with control vector. RLU, relative luminometer units.

PE-stimulated ANP and MLC Responses Do Not Require PKC Activation-- PLC activation generates two partially independent signaling pathways, one initiated by sn-1,2-diacylglycerol generation and activation of various isoforms of PKC and the other initiated by Ins(1,4,5)P₃ and inositol phosphate metabolites. To examine the contribution of PKCs to PE-induced hypertrophic signaling, cells were treated with the selective PKC inhibitor bisindolylmaleimide (1 µM). Isolated NCMs were transfected with ANP- or MLC-luciferase constructs and subsequently treated with PE (50 µM) with or without bisindolylmaleimide (1 µM). As shown in Fig. 3, inhibition of PKCs with bisindolylmaleimide did not significantly reduce signaling from PE to either ANP or MLC reporter genes. However, expression of both ANP- and MLC-luciferase was increased by direct activation of PKCs by phorbol 12-myristate 13-acetate (PMA, 10 µM), although the stimulation was less than with PE, and bisindolylmaleimide (1 µM) was inhibitory. The inactive isomer of bisindolylmaleimide, bisindolylmaleimide 5, was ineffective in reducing the PMA response, attesting to the specificity of the inhibition (Fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Inhibition of PKC reduces AP-1 responses, but not the ANP and MLC responses, to PE. NCMs were transfected with ANP-, MLC-, or 2XTRE (AP-1)-luciferase constructs and subsequently stimulated with PE (50 µM) or PMA (1 µM) together with bisindolylmaleimide 1 (active) or bisindolylmaleimide 5 (inactive). Luciferase activity was assayed 40 h later. Values shown are luminometer units per unit of -galactosidase, mean ± S.E. of triplicate estimations. Black bars, no inhibitor; gray bars, bisindolylmaleimide 1; open bars, bisindolylmaleimide 5. *, p < 0.01 relative to no additions. , p < 0.01 relative to PMA control. The experiment was performed three times. RLU, relative luminometer units; NA, no additions.

Another series of experiments was performed using a reporter construct encoding AP-1 response elements (2XTRE-luciferase) (34). Both PE (50 µM) and PMA (10 µM) increased AP-1 activity as indicated by increased luciferase activity, although in contrast to the ANP and MLC responses, PE was relatively weak compared with PMA. Responses to both effectors were inhibited by bisindolylmaleimide (1 µM) (Fig. 3). Thus, PE-stimulated activation of AP-1 response elements requires PKC activity, whereas transcriptional activation of ANP and MLC by PE is PKC-independent in this model (35).

Metabolism of Ins(1,4,5)P₃ Does Not Reduce ANP or MLC Responses to PE-- The finding of a requirement for PLC activity without PKC involvement suggests a role for the other arm of the PLC response, the inositol phosphates. To investigate the possible role of Ins(1,4,5)P₃ in the signaling pathways that link ₁-adrenergic receptor activation to increased expression of ANP and MLC, Ins(1,4,5)P₃-metabolizing enzymes were overexpressed, and responses to PE were evaluated. Pathways involved in the metabolism of Ins(1,4,5)P₃ are shown in Fig. 4. Overexpression of the enzyme that phosphorylates Ins(1,4,5)P₃ to Ins(1,3,4,5)P₄, Ins(1,4,5)P₃ 3-kinase (Fig. 4, enzyme 1), did not cause any change in ANP or MLC expression in the presence or absence of PE (Fig. 5, left panels). This argues against a major role for Ins(1,4,5)P₃ or InsPs derived from Ins(1,3,4,5)P₃, including Ins(1,3,4)P₃, and its metabolites. Overexpression of an enzyme that dephosphorylates Ins(1,4,5)P₃ to Ins(1,4)P₂, the 43-kDa type 1 Ins(1,4,5)P₃ 5-phosphatase (Fig. 4, enzyme 2), on the other hand, increased PE-stimulated responses (Fig. 5, right panels). The finding that increased dephosphorylation of Ins(1,4,5)P₃ actually stimulated ANP and MLC responses could be interpreted as either Ins(1,4,5)P₃ or the alternative substrate Ins(1,3,4,5)P₄ being inhibitory. However, the lack of effect of overexpression of the Ins(1,4,5)P₃ 3-kinase, which reduces Ins(1,4,5)P₃ while increasing Ins(1,3,4,5)P₄, argues against both possibilities.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Inositol phosphate metabolism. Substrates and products of enzymes used in the current study are shown. 1, Ins(1,4,5)P3 3-kinase; 2, Ins(1,4,5)P3 5-phosphatase; 3, INPP.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   ANP and MLC responses to PE are not reduced by increased metabolism of Ins(1,4,5)P3. NCMs were transfected with ANP- (upper panels) or MLC-luciferase (lower panels) together with plasmids encoding Ins(1,4,5)P3 3-kinase (IP3kinase) or Ins(1,4,5)P3 5-phosphatase (IP3Pase) as indicated. PE (50 µM) was added, and luciferase activity was measured after 40 h. Values shown are luminometer units per unit of -galactosidase activity, mean ± S.E. of triplicate estimations. The experiment was performed three times with similar results. Open bars, no additions; black bars, PE. *, p < 0.01 relative to no additions. , p < 0.01 relative to control vector. RLU, relative luminometer units.

Metabolism of Ins(1,4)P₂ Reduces ANP and MLC Responses to PE-- Another possible explanation for the stimulatory effect of overexpression of an enzyme that dephosphorylates Ins(1,4,5)P₃ to Ins(1,4)P₂ is that Ins(1,4)P₂ itself enhances hypertrophic signaling. To test this possibility, cardiomyocytes were transfected with the enzyme that dephosphorylates Ins(1,4)P₂ to Ins(4)P₁, INPP (Fig. 4, enzyme 3), together with ANP-luciferase or MLC-luciferase constructs. Overexpression of INPP inhibited both ANP and MLC responses to PE in a dose-dependent manner as shown in Fig. 6A. To examine the specificity of the observed inhibitory effect of INPP, transcription from ANP and MLC promoters was stimulated by overexpressing constitutively active JNK-1 together with c-Jun. Increased transcription was not inhibited by INPP. This shows that the inhibitory action of INPP targets upstream signaling pathways involved in the hypertrophic response and that INPP overexpression is not generally toxic to the cells. To further establish the specificity of the effect of INPP on hypertrophic signaling, we transfected NCMs with full-length antisense INPP and examined effects on ANP responses to 50 µM PE. Antisense INPP reduced INPP content in CHO cells and increased ANP transcription in the presence and absence of PE, providing further evidence that activity of this enzyme can regulate signaling pathways culminating in ANP transcription (Fig. 6B).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   A, ANP and MLC responses to PE are reduced by overexpressing INPP, but the response to activated c-Jun is not. NCMs were transfected with ANP- or MLC-luciferase constructs together with CMV--galactosidase and the indicated amount of pCR3.1-FLAG-INPP. Where indicated the cells were also transfected with c-Jun and a constitutively active JNK (1.2 µg of DNA for each). Total DNA was maintained at 4.8 µg with pCR3.1-CAT. PE (50 µM) was added, and luciferase activity was measured after 40 h. ANP and MLC values shown are luminometer units per unit of -galactosidase activity. All values are mean ± S.E. of triplicate estimations. The experiment was performed three times with similar results. *, p < 0.01 relative to no additions. , p < 0.01 relative to pCR3.1-CAT control. Open bars, no additions; black bars, PE; gray bars, c-Jun/JNK. B, ANP responses are enhanced by expression of antisense INPP. NCMs were transfected with ANP-luciferase, CMV--galactosidase, and pCR3.1-antisense INPP (1.6 µg of DNA) (anti) or pCR3.1-CAT (cont). The experiment and analysis were performed as described above. Open bars, no additions; black bars, PE. Expression of INPP in CHO cells transfected with antisense INPP using anti-INPP antibody as described in the legend to Fig. 1 is shown beneath. C, overexpression of INPP is not cytopathic. NCMs were transfected with pCR3.1-FLAG-INPP together with pEGFP-Cl to identify transfected cells. After 40 h, cells were photographed under phase contrast plus fluorescence microscopy (EGFP) (a), fluorescence only (b), and phase contrast only (c). Shown is a representative field. The experiment was performed four times with more than 20 fields examined per experiment. The bar represents 50 µm. RLU, relative luminometer units.

To further examine possible cytotoxic effects of INPP overexpression, we transfected NCMs with pCR3.1-FLAG-INPP together with pEGFP-Cl so that transfected cells could be identified by EGFP fluorescence. Fluorescent cells were viable, and there was no indication of cell rounding or cytotoxicity (Fig. 6C).

Metabolism of Ins(1,4)P₂ Reduces rDNA Transcription-- Increases in ribosomal gene transcription and ribosome biogenesis are a prerequisite for PE-mediated cardiac hypertrophy (26, 27). In experiments similar to those described above, the effect of overexpression of INPP on rDNA transcription was assessed using pSMECAT, an accurate reporter for rDNA (26). NCMs were transfected with pCR3.1-FLAG-INPP together with pSMECAT and subsequently stimulated with PE for 40 h. Overexpression of INPP reduced pSMECAT activity in response to PE, as shown in Fig. 7, suggesting a role for INPP in signaling pathways directly related to cell growth.

View larger version (9K): [in this window] [in a new window]   Fig. 7.   Overexpression of INPP reduces rDNA transcription in response to PE. NCMs were transfected with pSMECAT (45 S ribosomal gene reporter) and subsequently treated with PE (50 µM). CAT activity was measured 40 h later. Values shown are CAT activity in phosphorimaging units/mg of protein, mean ± S.E. of triplicate determinations. The experiment was performed three times. Open bars, control cells; black bars, PE-treated cells. *, p < 0.01 relative to control cells. , p < 0.01 relative to control vector.

Hypertrophied Myocytes Contain Heightened Ins(1,4)P₂ Levels-- The inhibitory activity of INPP suggested that its substrate, Ins(1,4)P₂, was in some way involved in the hypertrophic response. To provide further evidence for this, we looked for evidence of perturbed INPP activity in hypertrophied myocytes by measuring the levels of the substrate of INPP, Ins(1,4)P₂, relative to the product, Ins(4)P₁. NCMs were treated with PE to induce hypertrophy in medium containing [³H]inositol (15 µCi/ml) to label the inositol phospholipids. [³H]Inositol-labeled cells, control and hypertrophied, were subsequently stimulated for 20 min with 100 µM NE in the presence of 10 mM LiCl (to inhibit breakdown of the isomers of InsP₁) as described under "Experimental Procedures." [³H]InsPs were extracted, and the different isomers were separated and quantified. As shown in Fig. 8, hypertrophied myocytes contained heightened levels of [³H]Ins(1,4)P₂ and showed increased ratios of Ins(1,4)P₂ to Ins(4)P₁ suggesting reduced INPP activity in this hypertrophic model.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Hypertrophied NCMs contain heightened levels of Ins(1,4)P2. NCMs were [3H]inositol-labeled and treated with PE (50 µM) for 40 h. [3H]Inositol-labeled hypertrophied and control cells were stimulated with 100 µM NE for 20 min in the presence of propranolol and LiCl. [3H]InsPs were extracted and quantified. A, anion exchange HPLC analysis showing Ins(1,4)P2 content of control and hypertrophied NCMs. B, content of Ins(1,4)P2 in control and hypertrophied NCMs expressed as the ratio of Ins(1,4)P2 to Ins(4)P1 in percent. Gray bars, control NCMs; black bars, hypertrophied NCMs. Values shown are mean ± S.E. from a single experiment performed in triplicate. The experiment was performed three times with similar results. *, p < 0.01 relative to non-hypertrophied cells.

INPP Activity Influences Contraction-induced Hypertrophy in NCMs-- As PE itself is a direct activator of InsP generation, chronic treatment may have influenced InsP metabolism. For this reason, we sought to repeat the above studies using the contraction-induced hypertrophic model of neonatal cardiomyocyte hypertrophy as described under "Experimental Procedures." [³H]Inositol-labeled contracting (hypertrophied) and contraction-arrested (non-hypertrophied) cells were stimulated with 100 µM NE for 20 min, and [³H]InsPs was extracted, separated, and quantified. [³H]Ins(1,4)P₂ content was substantially higher in the contracting, hypertrophied myocytes (Fig. 9A), and the ratio of Ins(1,4)P₂ to Ins(4)P₁ was heightened (Fig. 9B).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Ins(1,4)P2 levels are increased in NCMs hypertrophied by spontaneous contraction, and overexpression of INPP reduces hypertrophic responses in this model. A and B, [3H]inositol-labeled hypertrophied (Contracting) and control (Arrested) cells were stimulated with 100 µM NE for 20 min in the presence of propranolol and LiCl. A, [3H]InsPs were extracted and quantified by HPLC. B, content of Ins(1,4)P2 in control and hypertrophied NCMs expressed as the ratio of Ins(1,4)P2 to Ins(4)P1 in percent. Values shown are mean ± S.E., n = 3. *, p < 0.01 relative to non-hypertrophied cells. C and D, NCMs were plated at high density and allowed to contract spontaneously (hypertrophied cells) or arrested with 50 mM KCl (non-hypertrophied cells) and were transfected with ANP-luciferase or pSMECAT as described under "Experimental Procedures." After 40 h, luciferase and CAT activities were measured. Gray bars, arrested NCMs; black bars, contracting NCMs. All values are mean ± S.E. of triplicate estimation and all experiments were performed three times. *, p < 0.01 relative to arrested cells. , p < 0.01 relative to control vector. RLU, relative luminometer units.

As described previously, contracting NCMs showed heightened expression of ANP and increased rDNA transcription (26, 27). In experiments similar to those described above, INPP was overexpressed in arrested or contracting NCMs, and markers of ANP expression (ANP-luciferase) or rDNA transcription (pSMECAT) were measured. As shown in Fig. 9, C and D, overexpression of INPP reduced both the ANP and rDNA responses induced by spontaneous contraction. Thus, the inhibitory activity of INPP is not restricted to PE-induced hypertrophy.

Hypertrophied Mouse Hearts in Vivo Contain Heightened Levels of Ins(1,4)P₂-- Mice were subjected to thoracic aortic constriction (TAC) to induce pressure overload hypertrophy or to sham operation as described previously (28). 6 weeks after TAC, hearts were removed, and left ventricles were dissected, mounted in organ baths, and labeled with [³H]inositol. Labeled strips were subsequently stimulated with 100 µM NE for 20 min, [³H]InsPs were extracted, and the individual isomers were separated and quantified as described previously (36, 37, 28). As described in cell models of hypertrophy, hypertrophied left ventricle contained higher levels of [³H]Ins(1,4)P₂ compared with ventricles from sham-operated animals (Fig. 10A), and the ratio of Ins(1,4)P₂ to Ins(4)P₁ was heightened (Fig. 10B). Thus, the metabolism of Ins(1,4)P₂ appears to be reduced in this model of hypertrophy in vivo, and when considered with the in vitro data, decreased activity of INPP is a common feature of diverse forms of cardiomyocyte hypertrophy.

View larger version (11K): [in this window] [in a new window]   Fig. 10.   Hypertrophied mouse hearts contain reduced Ins(1,4)P2. Mouse hearts were hypertrophied by thoracic aortic constriction and harvested 6 weeks after surgery. Left ventricle strips were labeled with [3H]inositol and subsequently stimulated with 100 µM NE for 20 min. A, [3H]InsPs were extracted and quantified by HPLC. B, content of Ins(1,4)P2 in control and hypertrophied ventricles expressed as the ratio of Ins(1,4)P2 to Ins(4)P1. Gray bars, sham; black bars, TAC. Values shown are mean ± S.E., n = 6. *, p < 0.01 relative to sham-operated animals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ventricular myocytes increase in size under pathological conditions in vivo and in vitro. Such hypertrophic growth is associated with increased expression of a number of genes, including ANP and MLC, and these gene are often used as "markers" for pathophysiological hypertrophic responses. The importance of pathways downstream of G_q in initiating hypertrophic growth in cardiomyocytes is well established in in vivo studies (38) as well as in experiments using rat neonatal cardiomyocyte models (4). Currently the only well established targets for activated G_q are the -isoforms of PtdIns-specific PLC (5). As PLC activation generates both inositol phosphates and sn-1,2-diacylglycerol, either PKC family members, inositol phosphates, or both might be involved in activating downstream hypertrophic signaling molecules. There is currently substantial evidence for PKC involvement in aspects of the hypertrophic response (3), but the possible involvement of the inositol phosphate arm of the pathway has been less thoroughly investigated.

In the current experiments, we found that the PKC inhibitor bisindolylmaleimide inhibited ANP and MLC responses to PMA but was unable to prevent responses to ₁-adrenergic receptor stimulation, suggesting a lack of requirement for PKC in this particular aspect of hypertrophy. This is consistent with findings in a recent study where modifying the activity of PKC while altering some aspects of cardiac growth responses to G_q overexpression in vivo did not perturb the increased expression of either ANP or MLC (7). Bisindolylmaleimide was able to inhibit PE-induced increases in transcription from an AP-1 reporter construct and also inhibited ANP and MLC responses to PMA. This shows that bisindolylmaleimide is able to access PKC isoforms effectively in NCMs under our experimental conditions. The PMA data also show that activation of at least one of the isoforms of PKC can increase ANP and MLC transcriptions. However, it also implies that this isoform either is not activated by PE or that PE stimulation of this isoform is exactly balanced by activation of an isoform that inhibits the response. The apparent lack of necessity for PKC in PE-dependent activation of ANP and MLC gene expression does not preclude a role for PKC in other aspects of the hypertrophic response to PE, including increases in cell size.

The lack of involvement of PKC in signaling pathways linking PE to ANP and MLC promoters pointed to a role for the inositol phosphate arm of the pathway. Given its well established association with Ca²⁺, Ins(1,4,5)P₃ was the most likely contender (12, 39). Increased cytosolic Ca²⁺ could contribute to hypertrophic signaling by activating conventional PKC isoforms and by stimulating Ca²⁺-activated phosphatases such as calcineurin (40) or calmodulin-activated kinase IV (10). However, overexpression of Ins(1,4,5)P₃-metabolizing enzymes did not reduce PE-stimulated increases in ANP or MLC transcription. Overexpression of Ins(1,4,5)P₃ 5-phosphatase actually increased transcription of both reporter genes, which might suggest an inhibitory role for one of its two substrates, Ins(1,4,5)P₃ or Ins(1,3,4,5)P₄. However, overexpression of Ins(1,4,5)P₃ 3-kinase, which metabolizes Ins(1,4,5)P₃ to Ins(1,3,4,5)P₄ and would thus be expected to decrease Ins(1,4,5)P₃ and increase Ins(1,3,4,5)P₄, had no effect on signaling, arguing that neither of these InsPs is important in signaling pathways culminating in ANP or MLC transcription.

The other possible explanation for the enhanced ANP and MLC responses in cells overexpressing Ins(1,4,5)P₃ 5-phosphatase is that the product of the Ins(1,4,5)P₃ 5-phosphatase, Ins(1,4)P₂, is stimulatory. In agreement with this, overexpression of the enzyme primarily responsible for metabolizing Ins(1,4)P₂, INPP, inhibited the responses (Figs. 6, 7, and 9). In addition to Ins(1,4)P₂, INPP removes the 1-phosphate from Ins(1,3,4)P₃ (19), but our data suggest that increased Ins(1,3,4)P₃ is unlikely to be responsible for the observed inhibitory action of INPP as it is generated by pathways initiated by Ins(1,4,5)P₃ 3-kinase, and overexpressing this enzyme had no effect on ANP and MLC responses. However, it remains possible that the hydrolysis product of INPP, Ins(4)P₁, is inhibitory to hypertrophic signaling rather than the substrate, Ins(1,4)P₂, being stimulatory.

The central role of Ins(1,4,5)P₃ in regulating intracellular Ca²⁺ responses in non-excitable cells is well established (41), although its functional importance in excitable tissues such as heart is less clear (12). Functional roles have also been ascribed to a number of other InsPs. Ins(3,4,5,6)P₄ can regulate Cl channels in some cell types (42), and a role in transcriptional regulation has also been suggested (43). InsP₆ has been assigned roles in nuclear export of mRNA (44) and in repair of DNA double strand breaks (45). In the case of Ins(1,4)P₂, a role in cellular growth responses was reported in studies where exogenously expressed INPP localized to the nucleus and reduced DNA synthesis (46). Furthermore, Ins(1,4)P₂ itself has been shown to activate DNA polymerase- (47). However, given that postnatal cardiomyocytes are terminally differentiated and do not undergo substantial cell division, such effects on DNA synthesis are unlikely to be involved in the antihypertrophic action of INPP demonstrated in these experiments. In addition to reducing transcription from ANP and MLC promoters, overexpression of INPP was found to inhibit rDNA transcription. As increased ribosome synthesis has previously been shown to be a prerequisite for both PE- and contraction-mediated hypertrophic growth of cardiomyocytes (27, 48), this suggests that Ins(1,4)P₂ may be involved in pathways regulating cellular growth in addition to its effect on ANP and MLC transcription.

The inhibitory effect of overexpressing INPP on transcription of genes associated with hypertrophic growth pointed to a role for Ins(1,4)P₂ in hypertrophic signaling pathways. In keeping with this, heightened levels of Ins(1,4)P₂ were observed not only in PE-induced hypertrophy but also in hypertrophy associated with contraction and more importantly in hypertrophied left ventricle in vivo. The finding of increased Ins(1,4)P₂ relative to Ins(4)P₁ in three different hypertrophic models implies that reduced activity of INPP is a common feature of hypertrophy. While such changes might be secondary to the hypertrophy, the finding that increased INPP expression can reduce at least some components of the hypertrophic response suggests that Ins(1,4)P₂ has a functional role. In contrast to these models of hypertrophy where Ins(1,4)P₂ levels are increased, we have previously reported reduced levels of Ins(1,4)P₂ in heart tissue from rats chronically fed diets enriched in n-3 fatty acids (49). We have also observed an acute decrease in Ins(1,4)P₂ in cardiomyocytes, in isolated atria, and in perfused rat hearts under conditions of ischemia or hypoxia (36, 50). Lowered Ins(1,4)P₂ implies increased activity or expression of INPP as opposed to the situation in hypertrophy where activity appears to be decreased.

Previous studies from our laboratory have shown that PLC activation via ₁-adrenergic receptors in neonatal rat cardiomyocytes generates both Ins(1,4,5)P₃ from PtdIns(4,5)P₂ and Ins(1,4)P₂ from PtdIns(4)P₁ (14). Given that Ins(1,4,5)P₃ is potentially arrhythmogenic in heart, the generation of Ins(1,4)P₂ might serve primarily to minimize changes in Ins(1,4,5)P₃ while maintaining the ability to produce diacylglycerol and thus to regulate PKC. The current experiments demonstrate that Ins(1,4)P₂ has a function of its own in signaling pathways in cardiomyocytes, and thus direct generation of Ins(1,4)P₂ may be an important aspect of cardiac growth responses.

	ACKNOWLEDGEMENTS

We gratefully acknowledge Prof. Christina Mitchell (Monash University) for the plasmid expressing Ins(1,4,5)P₃ 5-phosphatase, Dr. Christophe Erneux (Free University of Brussels, Belgium) for the Ins(1,4,5)P₃ 3-kinase plasmid, Dr. N. Dhanasekaran (Temple University, Philadelphia, PA) for the c-Jun and JNK plasmids, and Dr. Walter Thomas (Baker Institute) for the AT-1_A plasmid. INPP was originally cloned in the laboratory of Prof. Bruce Kemp (St. Vincent's Institute for Medical Research, Melbourne, Australia). We thank Bronwyn Kenney for technical assistance.

	FOOTNOTES

^* This work was supported by the Australian National Health and Medical Research Council. Some of this work was performed under the auspices of an international exchange program between the NHLBI, National Institutes of Health and the Baker Research Institute (granted to E. A. W. and J. H. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Baker Inst., PO Box 6492, St. Kilda Rd. Central, Melbourne, 8008, Victoria, Australia. Tel.: 61-3-85321255; Fax: 61-3-85321100; E-mail: liz.woodcock@baker.edu.au.

^§ Present address: The Center for Molecular Cardiology, Dept. of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032.

Published, JBC Papers in Press, April 3, 2002, DOI 10.1074/jbc.M110405200

	ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; ANP, atrial natriuretic peptide; MLC, myosin light chain-2; INPP, inositol polyphosphate 1-phosphatase; Ins, inositol; PKC, protein kinase C; InsP, inositol phosphate; PtdIns, phosphatidylinositol; NCM, neonatal cardiomyocyte; CMV, cytomegalovirus; JNK, c-Jun NH₂-terminal kinase; CAT, chloramphenicol acetyltransferase; CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; PE, phenylephrine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NE, norepinephrine; WT, wild type; PMA, phorbol 12-myristate 13-acetate; EGFP, enhanced green fluorescent protein; TAC, thoracic aortic constriction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES