Departments of 1Cell Biology and Anatomy and 2Medicine, New York Medical College, Valhalla, New York
Submitted 8 July 2004 ; accepted in final form 20 November 2004
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ABSTRACT |
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pulmonary hypertension; caveolin-1; pyrrolizidine alkaloids
The pyrrolizidine alkaloids need to be bioactivated in the liver to pyrrolic derivatives via the cytochrome P450 system (for review, see Refs. 22, 3032, 67). Thus, although MCT by itself produced megalocytosis in MCT-treated primary human hepatocyte cultures (16), it is the monocrotaline pyrrole (MCTP) that is active in producing megalocytosis in other cell types, including endothelial cells (for reviews, see Refs. 22, 28, 3032, 67). Because the half-life of MCTP in aqueous buffers is only 3 s (30) and a single administration of MCT, which is cleared in <24 h, is effective in causing PH 1014 days later (20, 28, 33), the in vivo effect of pyrrolizidine alkaloids has come to be viewed as a "hit-and-run" mechanism. This hit-and-run characteristic has been extensively validated in cell culture (16, 22, 28, 46, 67).
Although megalocytosis of both endothelial cells and hepatocytes involves marked cellular enlargement with nuclear enlargement and blocked/abnormal mitoses, there is a distinct difference in the effect on DNA synthesis in the two cell types. In hepatocytes, both in vivo and in cell culture, megalocytosis is characterized by an inhibition of DNA synthesis (3, 17, 40, 58), with a block in the ability of such cells to enter M (30). However, in pulmonary arterial endothelial cells (PAEC), both in vivo and in cell culture, megalocytosis is accompanied by stimulation of DNA synthesis, but still with a block in G2/M, resulting in enlarged cells that are hypertetraploid (34; for reviews, see Refs. 22, 28, and 67). However, subsets of both megalocytotic hepatocytes and endothelial cells show continuing DNA synthesis (3, 22, 28, 58, 67).
Despite >60 years having elapsed since the first observation of megalocytosis by Harris and colleagues (13, 14) and >40 years having passed since it was first observed in cell culture (16), the subcellular basis for megalocytotic transformation has not been elucidated. Although the MCT-induced PH model has been extensively studied during the past four decades, the cellular mechanisms contributing to megalocytosis of PAECs are only now beginning to be explored (for reviews, see 22, 28, 43, 61, 67). This laboratory (28) proposed recently that MCT induced a disruption of caveolin (Cav)-1/raft function in PAEC, resulting in widespread alterations in cell signaling, including stimulation of promitogenic STAT3 and ERK1/2 signaling. However, the mechanisms underlying the block in cell cycle traversing to M have not been elucidated.
In the present study, we recapitulated MCTP-induced megalocytosis in cultures of bovine PAEC, extended these observations to cultured human Hep3B hepatocytes, human pulmonary type II-like alveolar epithelial cells (A549), and human pulmonary arterial smooth muscle cells (PASMC), and investigated the subcellular mechanism involved. The data suggest a unifying mechanism, which we term the "Golgi blockade" hypothesis, which appears to account for disruptions of Cav-1 protein trafficking and altered cell signaling as well as for a block in progression to mitosis.
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MATERIALS AND METHODS |
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For use in cell culture, MCTP was prepared from MCT (purchased from TransWorld Chemicals, Rockville, MD) using the procedure of Mattocks et al. (31). With the use of mass analysis, 3050% of the input MCT was converted to the pyrrolic derivative (data not shown). MCTP was stored in small aliquots in dimethylformamide (DMF) at 80°C, diluted to the required concentration in DMF just before use, and added directly to the cultures with gentle swirling. Control cultures received an equivalent volume of DMF. Recombinant human IL-6 was purchased from R & D Systems (Minneapolis, MN).
DNA synthesis. Respective cultures in six-well plates were pulsed with bromodeoxyuridine (BrdU, 10 µM; Sigma-Aldrich, St. Louis, MO) for 20 min, followed by methanol-acetone fixation (22, 37, 67). Nuclei were visualized (in blue) by staining with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Pierce Biotechnology, Rockford, IL), and BrdU incorporation was visualized by performing immunofluorescence imaging using anti-BrdU monoclonal antibody (MAb; Sigma-Aldrich) and rhodamine-tagged secondary antibody (Molecular Probes, Eugene, OR) using the respective manufacturer's protocols. The BrdU labeling index represents the fraction (in percent) of DAPI-stained nuclei showing BrdU labeling.
Double-labeled immunofluorescence studies.
PAEC in six-well plates were fixed with methanol-acetone (45), and double-labeled immunofluorescence studies were performed as reported previously (28) in various combinations using the respective antibodies for endothelium-selective Cav-1 [rabbit polyclonal antibody (PAb) sc-894], the cis-Golgi marker GM130 (murine MAb) and the protein kinase C
-isoform (PKC-
; rabbit PAb) using corresponding AlexaFluor 488-, AlexaFluor 594-, Cy3-, or rhodamine-tagged secondary antibodies (Molecular Probes). Controls included competition with Cav-1 peptide (sc-894 P) and nonimmune immunoglobulins (murine MAb and rabbit PAb). For microtubule and actin display, the cultures were washed with warm (37°C) phosphate-buffered saline (PBS), fixed in 3.7% warm formaldehyde in PBS for 30 min, permeabilized with 0.1% Triton X-100 for 15 min, and then immunostained using anti-
-tubulin MAb or phalloidin-rhodamine respectively (both from Molecular Probes). Fluorescent display of chromosomal DNA was visualized using DAPI staining. Images were collected using a MRC 1024 ES (Bio-Rad) confocal microscopy system or a Leitz epifluorescence microscopy system. All data in each experiment were collected at identical image settings.
Preparation of detergent-resistant membrane rafts. Triton X-100-resistant (0.05%) membrane rafts were prepared from PAEC using equilibrium sucrose density flotation according to the method of Lafont and Simons (19) as modified by members of our laboratory (28, 55, 56). Typically, seven fractions were collected from each gradient, corresponding to the visible band at the very top (fraction 1), just above the 13/43% sucrose interface (fraction 2), at the 13/43 interface (fraction 3), just below the 13/43 interface (fraction 4), at the 43/60 interface (fraction 5), an aliquot from the 60% loading region (fraction 6), and the pellet (fraction 7) (see Fig. 3A). Fractions 2, 3, 4, 5, and 7 were diluted in cold Triton-solubilizing buffer, resedimented at 15,000 g for 20 min, and resuspended in 200 µl of solubilizing buffer. Aliquots were assayed for the plasma membrane marker 5'-nucleotidase (Sigma) and for various proteins using SDS-PAGE and Western blotting. Members of our laboratory have previously characterized these fractions extensively (see Refs. 28, 55, 56); fraction 3 is the typical low-density, detergent-resistant membrane raft fraction.
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DNA gel shift assays. STAT-specific DNA-binding activity was assayed using the m67 mutant serum-inducible element (SIE) oligonucleotide derived from the c-fos promoter (purchased from Santa Cruz Biotechnology, Santa Cruz, CA) as described previously (38, 55, 56). Routinely, multiple exposures of each autoradiogram were obtained on Kodak XAR5 film to be within the linear range of exposure of the film. The identification of the serum-inducible factor (SIF)-A band as STAT3 homodimer was confirmed using anti-STAT3 supershift assays (see Refs. 38, 55, 56).
Immunopanning using protein A magnetic beads. Protein A magnetic beads were purchased from New England Biolabs (Beverly, MA) and used in immunopanning as described previously (56). Before being used in immunopanning experiments, the beads were blocked with 5% nonfat dry milk in PBS for 1 h and then washed three times with PBS. Aliquots of the respective fractions were adjusted to 0.05% Triton X-100 in (in mM) 25 Tris·HCl, pH 7.4, 150 NaCl, 5 EDTA, and 1 DTT (binding buffer), incubated overnight at 4°C with respective rabbit immunoglobulin, and then incubated again for 1 h at 4°C with the preblocked protein A magnetic beads. The magnetic beads were washed five times with binding buffer containing 0.05% Triton X-100 and then twice with binding buffer adjusted to 0.5% Triton X-100.
Antibodies and additional reagents.
Rabbit antibodies to endothelium-selective Cav-1 (sc-894) and the corresponding horseradish peroxidase (HRP)-conjugated antibody to STAT3, phospho-Ser-STAT3, Ser25-P-GM130, phospho-Thr/Tyr-ERK2, protein kinase C
-isoform (PKC-
), and nonimmune rabbit IgG, as well as murine MAb to cdc2 and nonimmune isotype-matched murine MAb, were purchased from Santa Cruz Biotechnology. Rabbit PAb to PY-STAT3 was obtained from Cell Signaling Technology (Beverly, MA). Murine MAb to STAT3, GM130, early endosomal antigen-1 (EEA-1), and immunoglobulin binding protein (BiP)/glucose regulated protein (GRP)78 were purchased from BD Biosciences Transduction Laboratories (San Diego, CA). Murine MAb to the
1-subunit of Na+-K+-ATPase was obtained from Novus Biologicals (Littleton, CO). The reagents 2-methoxyestradiol (2-ME) and nocodazole were purchased from Sigma-Aldrich.
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RESULTS |
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The double-labeled immunofluorescence analytical data in Fig. 4A provide additional evidence for trapping of Cav-1 in a GM-130-positive compartment of the Golgi in MCTP-treated PAEC. Figure 4B shows a series of controls that validate the double-labeled immunofluorescence analyses shown in Fig. 4A. Specifically, Fig. 4B shows the selective competition of Cav-1-specific immunostaining with the relevant Cav-1 peptide used as the immunogen. Figure 4C further verifies the specificity of the respective secondary antibodies used by illustrating that PKC- (39) and GM130 are largely in distinct compartments in such cells, even though the respective anti-rabbit and anti-murine IgG secondary antibodies shown in Fig. 4C were the same as those shown in Fig. 4, A and B.
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The immunopanning data in Fig. 5A (top) confirm the previously reported loss of Cav-1 from MCTP-treated PAEC from a P15 membrane fraction (which includes both plasma membrane and internal membranous organelles) and the association between Cav-1 and STAT3 and with PY-STAT3 in these membranes. Unexpectedly, there was increased association of cdc2 in these complexes and an increase in pGM130 in association with Cav-1 upon MCTP treatment (Fig. 5Ab, bottom). Figure 5, Ba and Bb, shows two alternative approaches to evaluate the phosphorylation state of GM130 in MCTP-treated PAEC. Figure 5Ba shows Western blots of whole cell extracts that demonstrate the reduction of Cav-1 upon MCTP treatment (second lane from left) and a detectable phosphorylation of GM130 (bottom). In Fig. 5Bb, the cell extracts were first immunopanned using anti-pGM130, and then the amount of GM130 pull-down was assayed by Western blotting. The figure thus shows increased pull-down of pGM130 upon treatment with MCTP alone as well as the increased association between pGM130 and cdc2 kinase (i.e., the kinase that phosphorylates GM130).
The increased Ser25 phosphorylation of GM130 in MCTP-treated PAEC was unexpected and suggests that in such cells the cell cycle might be blocked at a step subsequent to Ser phosphorylation of GM130 but before entry into M. This possibility was investigated in experiments in both PAEC and Hep3B cells in which the effect of MCTP on the entry of cells into M was studied using 2-ME. Figure 6 shows that 2-ME alone, as expected (4, 25, 47, 53), markedly increased the number of cells in M (the rounded refractile cells). That these rounded cells represent cells specifically blocked in mitosis (in metaphase plate block) is confirmed by the data shown in Figs. 7, B and C, and 8 (also see Refs. 4 and 53). Importantly, the data in Fig. 7C show that these rounded cells are not undergoing apoptosis but can slowly progress through M and exit into the next cell cycle (also see Refs. 4 and 53). Treatment of PAEC and Hep3B cultures with MCTP inhibited entry of cells into M (Figs. 68). Similar observations were obtained in A549 and PASMC cultures (data not shown). Remarkably, Figs. 2 and 3, published in 1980 by Mattocks and Legg (32), showing an inhibition of entry of rat hepatocyte cell line into M (determined using a colcemid block and metaphase spread assay) upon pretreatment with dehydroretronecine, anticipated our present observations with 2-ME and MCT.
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Figure 7 shows that in a direct assay for Golgi fragmentation, 2-ME failed to induce Golgi fragmentation in MCTP-treated PAEC. This inhibitory effect was not secondary to effects of 2-ME on microtubule or actin organization per se (Fig. 8) and was specific for Golgi fragmentation in 2-ME-treated cells in that Fig. 9 shows that Golgi fragmentation induced by nocodazole (9, 59) was not affected in megalocytotic PAEC. However, Golgi reassembly after nocodazole fragmentation and washout was slowed (>90% of control cells showed reassembly by 120 min of washout, compared with only 30% of MCTP-treated cells), even though centromere and microtubule reassembly during the washout phase was equivalent (Fig. 9). Taken together, the data in Figs. 59 highlight a disruption in the mitosis sensor function of the Golgi organelle after MCTP treatment, despite adequate Ser25 phosphorylation of GM130.
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DISCUSSION |
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The period from 1942 to 1970 witnessed a great deal of interest in pyrrolizidine alkaloids, including MCT, for their effects on hepatocytes and their potential role in understanding hepatic carcinogenesis (for reviews, see Refs. 1, 2, and 17). Hepatocyte megalocytosis was extensively characterized both in vivo (1, 2, 7, 13) and in cell culture (3, 16, 58). The present report extends the observation of MCT-induced megalocytosis in culture to human hepatoma Hep3B cells and to human pulmonary alveolar epithelial cells and PASMCs.
During the past four decades, administration of the pyrrolizidine alkaloid MCT into juvenile male rats has been investigated intensively as a model for PH and right ventricular hypertrophy (20, 43). In this now well-established model of PH, MCT administered in a single subcutaneous injection is bioactivated in the liver to the pyrrolic derivative, and, in its first pass through the lung (the half-life of active pyrrole is 3 s; Ref. 30), it affects mainly the pulmonary arterial endothelium. There is evidence of endothelial lactate dehydrogenase leak and a pulmonary vascular leak within 2448 h (52, 66). Within 34 days, there is three- to fivefold stimulation of the DNA labeling index and megalocytosis of PAEC with consequent changes 1) on the abluminal side, leading to increased deposition of collagen and insoluble elastin, as well as migration and megalocytosis of smooth muscle cells; and 2) increased adhesiveness of the PAEC surface to cellular elements in the bloodstream (for review, see Ref. 28). Subsequent vascular remodeling eventually leads to irreversible clinical PH by 1014 days.
In the MCT rat model of PH we previously reported the disruption of endothelial cell Cav-1/raft function, hyperactivation of promitogenic STAT3 and ERK1/2 signaling, and increased DNA synthesis at the single-cell level, both in vivo and in cell culture, specifically in cells showing reduced Cav-1 and increased PY-STAT3 levels (28). Moreover, the prior observations of a loss of the endothelial cell angiopoietin-1 receptor Tie-2 (70) and that of platelet endothelial cell adhesion molecule-1 (28) in this model are consistent with disruption of Golgi trafficking and Cav-1/raft function. In addition, the low bioavailability of NO in the lung despite unchanged endothelial NO synthase (eNOS) amounts (29) is also consistent with a reduction in Cav-1 chaperone function, which is needed to deliver eNOS from the Golgi to the plasma membrane raft. Indeed, Golgi trapping of eNOS in endothelial cells leading to reduced NO bioavailability has been reported in a model of hypoxia-induced PH (35). The available data suggest that the subcellular location of the respective signaling molecules and receptors, whether trapped in the Golgi or efficiently chaperoned to the plasma membrane raft, critically affects the overall functional phenotype. MCT would be expected to disrupt the subcellular location of diverse signaling molecules by blocking trafficking through the Golgi, leading to widespread alterations in cell signaling in megalocytotic cells.
The equilibrium flotation data in Fig. 3 confirm the block in bulk trafficking through the Golgi: there is a shift in the density of membranes with the cis-Golgi marker GM130, together with Cav-1 and BiP to a lighter density (from fraction 5 to fractions 3 and 4) in PAEC exposed to MCTP 48 h earlier. The immunofluorescence data in Fig. 4 confirm the increased trapping of Cav-1 in the Golgi in MCTP-treated cells. Importantly, accumulation of hypo-oligomeric Cav-1 in a GM-130-positive light membrane fraction (see Fig. 3B, fraction 3, and the velocity sedimentation analyses after n-octyl-glucoside treatment in Fig. 6B of Ref. 28) is also consistent with trapping of incompletely assembled Cav-1 oligomers in the Golgi compartment.
The consequences MCTP-induced megalocytosis on promitogenic cell signaling are complex. While there is hyperactivation of PY-STAT3, there is a loss of PS-STAT3. Nevertheless, Tyr-Thr phosphorylation of ERK1/2 is enhanced, as is the Ser phosphorylation of GM130. MCTP treatment enhanced STAT3 DNA binding activity in the nucleus in both PAEC and Hep3B. However, recent reports have suggested that although STAT3 has a promitogenic effect in specific cell types (5, 6), it also can be inhibitory in others (51). Thus our observation that MCTP enhanced DNA synthesis in PAEC but inhibited that in Hep3B cells, which confirms data in the pyrrolizidine alkaloid literature (see Introduction), suggests that activation of the same transcription factor (in this case, STAT3) may have opposite effects in the two cell types. This is a question that requires additional investigation.
In terms of a block of cell cycle traverse, the observations that MCTP treatment inhibited fragmentation of the Golgi and entry into M induced by 2-ME in both PAEC and Hep3B cells and that reformation of the Golgi upon nocodazole fragmentation was slowed in megalocytotic cells reinforce the hypothesis that disruption of overall Golgi function is a significant consequence of MCTP exposure. Altered Golgi dynamics would be expected to lead to a failure to enter mitosis and the continued enlargement of cells despite ongoing macromolecular synthesis. Ser phosphorylation of the Golgi scaffolding protein GM130 by cdc2 kinase is thought to initiate a disruption of the ternary complex between GM130, p115, and giantin, resulting in the initiation of Golgi fragmentation, subsequent nuclear dissolution, and entry of the cell into mitosis (the mitosis sensor function of the Golgi) (12, 54, 59). We observed increased Ser phosphorylation of GM130 in complexes that contained cdc2 and Cav-1 in MCTP-treated cells (with the highest levels observed in cells that received both MCTP and 2-ME), and yet the cells did not proceed through to M, suggesting that MCTP may help to define a cell cycle checkpoint at a step subsequent to Ser phosphorylation of GM130 but before Golgi fragmentation and entry into M. The alternative possibility that MCTP-treated PAECs cycle through G1/S/G2/G1/S and bypass M cannot be excluded at present.
Numerous investigators have evaluated the biochemical targets covalently modified by MCTP. MCTP is known to alkylate DNA in bovine PAECs in culture within 1 h (see Refs. 67 and 68 and references therein). It has been thought that this covalent modification of DNA might account for the inhibition of cell cycle traverse (67, 68). However, there is a stimulation of the DNA labeling index in PAECs and not an inhibition. More recently, Lamé et al. (21) identified specific proteins responsible for catalyzing Cys-Cys bridges and protein oligomerization as targets for covalent derivatization by MCTP-protein disulfide isomerase (PDI) and its isoform, called GRP58/ER60/ERp57, and suggested that the Cys-X-X-Cys motif might be a hyperreactive target element. Remarkably, both PDI and GRP58/ER60/ERp57 are located within the lumen of the ER and the Golgi, and their loss of function would impair trafficking through these organelles. Because proper assembly and oligomerization of Cav-1 in the Golgi requires its lipid modification on three specific Cys residues, without which this plasma membrane scaffolding protein remains trapped in the Golgi (57), we suggest that covalent modification of Cys residues on target proteins, including Cav-1, may initiate the trafficking block in the Golgi. In addition, MCTP has been reported to derivatize the cytoskeletal protein actin covalently (61, 68). However, we did not observe altered microtubule or actin organization per se in cells treated with MCTP.
From a broader perspective, it is well established that plant and fungal products have specific effects on Golgi function, and such inhibitors are in widespread use as investigational probes for Golgi function. For example, swainsonine inhibits -mannosidase II in the Golgi (65), and brefeldin A causes fragmentation of the Golgi and helps identify a specific protein CtBP3/BARS responsible for driving fission of Golgi membranes during mitosis (8, 12, 54). That a pyrrolizidine alkaloid, australine, inhibited amyloglucosidase and glycoprotein processing in the Golgi at the biochemical level also has been elucidated (64). The present report adds MCTP to the list of plant products that affect specific Golgi function in mammalian cells.
Another recently elucidated example of a Golgi trapping mechanism leading to human disease has been demonstrated in mechanistic studies of mutations in polycystin species and the development of autosomal dominant polycystic kidney disease (9, 36, 44). The disease-causing mutations in polycystin-1 lead to trapping of this protein in the Golgi; subsequent Golgi dysfunction, including trapping of Cav-1 (see Fig. 3 in Ref. 9); and a proliferative response in the renal epithelium.
To summarize, the present data indicate that the long-investigated megalocytosis phenotype produced by pyrrolizidine alkaloids likely results from a blockade of Golgi function, with attendant alterations in cell signaling phenotype and cell cycle traverse (the Golgi blockade hypothesis).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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