Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4256-4260, May 1993 Cell Biology Low density - and high density lipoprotein-mediated signal transduction and exocytosis in alveolar type II cells (surfactant/phosphatidylinositol turnover/LDL receptors/ E/apolipoprotein A-I) TATYANA A. VOYNO_YASENETSKAYA*, LELAND G. DOBBS*t#, SANDRA K. ERICKSONt§, AND ROBERT L. HAMILTON*$ *Cardiovascular Research Institute, Departments of tMedicine and 1Anatomy, University of California, San Francisco, CA 94143-0130; and Veterans Affairs Medical Center, San Francisco, CA 94121 Communicated by John A. Clements, January 12, 1993 (received for review August 26, 1992)

ABSTRACT Low density (LDL) and high poproteins (7). HDL both delivers to cells and density lipoproteins (HDL) from serum stimulate signal- promotes its removal (8). HDL-mediated cholesterol delivery transduction pathways and exocytosis in rat alveolar type II occurs, in part, by a selective cellular uptake mechanism (9). cells. Both LDL and HDL stimulated primary cultures of type Although HDL functions in a passive capacity as a choles- II cells to secrete phosphatidylcholine (PtdCho), the major terol acceptor for plasma-membrane cholesterol, the trans- phospholipid component of pulmonary surfactant. The effects location ofcholesterol from intracellular compartments to the on secretion were preceded temporally by stimulation of inosi- plasma membrane requires the specific interaction of HDL tol phospholipid catabolism, calcium mobilization, and trans- with a high-affinity cell-surface receptor (10, location of protein kinase C from cytosolic to membrane 11). compartments. Heparin, which blocks the binding ofligands to In addition to transport, LDL and HDL affect a the LDL receptor, completely inhibited the effects of LDL on variety of cellular functions. In cultured endothelium and signal transduction and PtdCho secretion but did not inhibit the smooth muscle cells, both LDL and HDL induce prolifera- effects ofHDL. Unilamellar PtdCho liposomes the size ofnative tion (12) and stimulate synthesis of prostacyclin and pros- LDL had no effect on type II cells; however, PtdCho complexes taglandin E2 (13, 14). LDL increases glycosoaminoglycan containing either apolipoproteins E or A-I stimulated both secretion by human smooth muscle cells and fibroblasts (15). signal transduction and PtdCho secretion. LDL receptors were Activation of inositol phospholipid catabolism and calcium present in type II cell membranes by immunoblotting. In mobilization occurs during some LDL- and HDL-mediated contrast to findings with hepatic membranes, type II cells cellular events (16-19). exhibited two major bands of 130 kDa and 120 kDa and a minor Inositol phospholipid catabolism (20), calcium mobiliza- band at 230 kDa that also was present under reducing condi- tion (21, 22), and protein kinase C (PKC) (23) are linked to tions. These results are consistent with our hypothesis that the surfactant secretion in cultured type II cells; we therefore LDL-receptor pathway functions in vivo to deliver cholesterol investigated whether LDL and HDL stimulate signal trans- to type II cells and that this process is coupled to surfactant duction and surfactant secretion in type II cells. assembly and secretion via signal-transduction pathway(s). HDL elicits similar responses independent ofthe LDL receptor, suggesting that type II cells may use the selective uptake MATERIALS AND METHODS pathway to obtain cholesterol or that HDL triggers signal Materials. Dulbecco's modified Eagle's tissue culture me- transduction by mechanisms unrelated to lipid delivery. dium (DME H-16) and fetal calf serum were obtained from the University of California Cell Culture Facility (San Fran- Cholesterol is the second most abundant lipid component of cisco), rats were from Bantin & Kingman (Fremont, CA), pulmonary surfactant, composing from 10 to 25% (by mole phorbol 12-myristate-13-acetate (PMA) was from Calbio- fraction) of total surfactant lipid (1, 2). The function of chem, indo-lAM was from Molecular Probes, rabbit apoE cholesterol in surfactant has not been established; however, and apolipoprotein, A-I (apoA-I) were from BioDesign (New it is believed to facilitate spreading of dipalmitoylphosphati- York), L-a-lecithin was from Avanti Polar-, and dylcholine at the air-liquid interface in the lung, lowering [3H]choline, myo-[H]inositol, [y32P]ATP, and 125I-labeled surface tension (1, 2). At least 80%o of lung cholesterol donkey anti-rabbit IgG were from Amersham. Other reagents appears to be derived from plasma lipoproteins (3, 4); most were from Sigma. We prepared fibronectin by using the of the cholesterol in lung surfactant also appears to originate method of Ruoslahti et al. (24). from lipoproteins (3, 5). Although it is well-established that Isolation and Culture of Alveolar Type II Cells. Alveolar alveolar type II cells, in addition to their important functions type II cells were isolated from adult rat lungs and cultured of lung repair and ion transport, synthesize and secrete as described (25). pulmonary surfactant (6), there is little information concern- Isolation ofLDL and HDL. LDL (density, 1.019-1.063) and ing the role of lipoprotein receptors in these cells. HDL (density, 1.063-1.21) were isolated from the plasma of Low-density (LDL) and high-density lipoproteins (HDL) different normocholesterolemic volunteers by ultracentrifu- are serum cholesterol-transporting molecules. When cells gation, according to the procedure of Havel et al. (26). require cholesterol, they increase de novo cholesterol syn- Lipoproteins were stored at 4°C and used within 3 weeks. No thesis and often up-regulate LDL receptors to obtain lipo- change in lipoprotein activity was seen during this period. protein cholesterol by receptor-mediated ofapo- lipoprotein B100- or (apoE)-containing li- Abbreviations: LDL, low density lipoprotein(s); HDL, high density lipoprotein(s); PtdIns, phosphatidylinositol; PtdCho, phosphatidyl- choline; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase The publication costs of this article were defrayed in part by page charge C; apoE and apoA-I, apolipoprotein E and A-I, respectively; InsP3, payment. This article must therefore be hereby marked "advertisement" inositol 1,4,5-trisphospfiate; [Ca2+]I, intracellular Ca2+ concentration. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 4256 Downloaded by guest on September 28, 2021 Cell Biology: Voyno-Yasenetskaya et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4257

The lipoproteins were dialyzed on the day of the experiment 2- A against 0.15 M NaCl/1 mM EDTA, pH 7.4. Protein content 1 was measured by the method of Lowry et al. (27). 0110. Preparation of Phosphatidylcholine (PtdCho)-ApoE or (I)0 8 a ATP -ApoA-I Complexes. Unilamellar PtdCho liposomes, diame- 0 6 ter =200 A, were prepared by using a French pressure cell, cn DL according to Hamilton et al. (28). Apolipoprotein complexes 4- LDL made from egg PtdCho unilamellar liposomes and rabbit 0 12 control apoE or apoA-I were prepared at a molar ratio of lecithin/ C. apolipoprotein of 300:1, as described by Pitas et al. (29). The 00 i i i apolipoprotein complexes used do not have a known stoi- 1 Time, h chiometry. oL Measurement of Surfactant Secretion. Surfactant secretion 2 B was measured by described methods (30), using [3H]PtdCho .6 1 HDL 0 0. as a marker for surfactant. la.I0J Determination ofInositol Phosolipid Turnover. Type II cells 0 (r I ILD were incubated in inositol-free Dulbecco's modified Eagle's I*IU) medium with myo[3H]inositol (2.5 ,uCi/ml; 1 Ci = 37 GBq) for 0 22 hr. The medium was removed, fresh medium containing 10 U,4-B mM LiCl was added, and cells were washed three times and 0 2- incubated for 30 min, before adding either control or test 0.T- solutions. Inositol phosphates were isolated, according to the 0 25 50 -g_m_ 100 method of Berridge et al. (31). [Lipoprotein] ,ug/ml PKC Assay. PKC activity was determined as phosphate transferred from ['y-32P]ATP to histone-III-S (32). Type II FIG. 1. LDL and HDL stimulate [3H]PtdCho secretion in alve- cells (5 x 106) cultured on 60-mm tissue culture dishes for 22 olar type II cells. [3H]choline-labeled type II cells were incubated hr were treated with with vehicle alone, LDL, HDL, or ATP. [3H]PtdCho was isolated lipoproteins, ATP, or PMA at 37°C for from media and cells, as described. Results represent means t SDs 5-15 min. Cells were broken and subfractioned into mem- of triplicate plates from three experiments, each with a different brane and cytosolic fractions and PKC activity was measured preparation oftype II cells. (A) Time course of [3H]PtdCho secretion as described by Sano et al. (23). PKC activity was calculated in response to 25 ,ug of LDL per ml, 25 jig of HDL per ml, or 0.1 mM as the difference in histone phosphorylation between samples ATP. (B) Dose-dependency ofLDL- and HDL-mediated [3H]PtdCho incubated with and without phosphatidylserine, diolein, and secretion. Different concentrations of either LDL or HDL were calcium. The total PKC activity recovered per unit of cells added, and the cells were incubated for 3 hr. was constant under all conditions. We measured PKC activ- ity induced by PMA (a positive control) at 10 min, the time (n = 3), and that for HDL was =15 ,g/ml (Fig. 1B, n = 3). of maximal PMA effect (23). We measured the effects of HDL and LDL were somewhat less potent secretagogues lipoproteins on PKC after 5 min because their effects on than ATP, a potent secretagogue for type II cells (37, 38). inositol trisphosphate (InsP3) generation were maximal at ATP (0.1 mM) increased the amount of [3H]PtdCho in the this time (see Fig. 2). medium 5- to 6-fold, whereas HDL or LDL at 25 ,ug/ml Calcium Measurement in Single Cells. The intracellular increased the amount of secreted [3H]PtdCho 2- to 4- or 1.5- Ca2+ concentration ([Ca2+]i) in single cells was quantitated to 2-fold, respectively. by the use of an ACAS model 570 laser cytometer. Type II Stimulation ofInositol Phospholipid Catabolism by HDL and cells were cultured on fibronectin-coated coverslips and then LDL. Both HDL and LDL induced a dose- and time-de- incubated with indo-lAM, as described (33). [Ca2+], was pendent accumulation of [3H]inositol phosphates in type II quantitated as described (33-35). cells (Fig. 2). The increase in both total inositol phosphates Determination of LDL Receptors in Type II Cells. LDL and in InsP3 ([3H]InsP3) occurred within 2 min (Fig. 2B, P < receptors were identified by SDS/PAGE ofeither whole cells 0.01 above control values). HDL was a more potent stimulus or cell membranes under both reducing and nonreducing of inositol phospholipid catabolism than was LDL (Fig. 2 A conditions, followed by transfer to nitrocellulose and immu- and B). Furthermore, the stimulatory effect of HDL on noblotting (36). Four rabbit antisera specific for the LDL accumulation of inositol phosphates was more prolonged receptor were used as primary antibodies. Three antisera, than that of LDL (Fig. 2). each raised against a different preparation of bovine adrenal Modulation of PKC Activity by HDL and LDL. We mea- LDL receptors, were provided by J. K. Boyles, University of sured the stimulation of PKC by evaluating translocation of California at San Francisco. One antiserum raised against PKC activity from cytosolic to fractions. In hepatic LDL receptors purified from ethynylestradiol- nonstimulated type II cells, up to 94% of PKC activity is treated rats was provided by R. J. Havel, University of recovered in the fraction (23, 39). Exposure of California at San Francisco. 1251-labeled donkey anti-rabbit alveolar type II cells to HDL and LDL resulted in an increase IgG was used as the secondary antibody; the bands were in membrane-associated PKC activity of 158 + 16% (n = 3) visualized by autoradiography. Hepatic sinusoidal mem- and 226 + 29% (n = 3), respectively. PMA, a positive control, brane-enriched fractions from of normal rats and from in a longer incubation than that used for HDL and LDL, rats treated with ethynylestradiol were used as reference induced a 400 ± 38% (n = 5) increase in membrane- standards (36). associated PKC activity (Fig. 3). Short-term activation ofthe cells either with lipoproteins or phorbol esters did not change the total cellular activity of PKC (data not shown). RESULTS Effects of HDL and LDL on [Ca2+11 in Single Cells. We Stimulatory Effect of LDL and HDL on PtdCho Secretion. measured [Ca2i]i in single type II cells using a laser cyto- Both HDL and LDL stimulated secretion of [H]PtdCho from meter. ATP (1 AM), which is known to increase [Ca2+1j in primary cultures of type II cells in a time- and dose- monolayers oftype II cells (40), was used as a positive control dependent manner (Fig. 1). The half-maximal concentration (Fig. 4 A and B). Both HDL and LDL at concentrations of25 of LDL required for PtdCho secretion was 5.3 + 2.4 ,g/ml ,g/ml increased [Ca2+], (Fig. 4 C-F). In experiments done Downloaded by guest on September 28, 2021 4258 Cell Biology: Voyno-Yasenetskaya et al. Proc. Natl. Acad. Sci. USA 90 (1993)

A InsP 800 ATP

ci 600 1( 5D 00 HDL .f0. 400 0 co0 200 D 20 (IO'l- ' C) 0 510 15 iO

_L O 400 0 's1w B InsP3 0c ' 0r- 300 0 ATP 0 -S 200,

CO, HD 100 LDL 200 0 50 100 150 200 Time, sec 1m5 iO 1,0 FIG. 4. Effect of LDL, HDL, and ATP on [Ca2+]i in single type Time, mmn II cells. Type II cells were isolated, loaded with indo-1, and fluo- rescence measurements were made, as described. (A) ATP (1 in FIG. 2. Time course of LDL and HDL stimulation of inositol AM) the presence of 2.2 mM Ca2+. (B) ATP (1 AM), medium without phospholipid turnover in alveolar type II cells. myo[3H]Inositol- Ca2+, containing 0.1 mM EGTA. (C) LDL at 25 pg/ml in the labeled type II cells were treated either with vehicle alone, LDL (25 presence of 2.2 mM Ca2+. (D) LDL at 25 .g/ml, medium without jAg/ml), or HDL (25 Ag/ml) for the times indicated. The accumula- Ca2+, containing 0.1 mM EGTA; then Ca2+ was added to 2.2 mM; tion of [3H]inositol phosphate (InsP) is shown in A and of [3H]InsP3 (E) HDL at 25 pg/ml in the presence of 2.2 mM Ca2+; (F) HDL at in B. The accumulation of [3H]inositol diphosphate (data not shown) 25 ug/ml, medium without Ca2+, containing 0.1 mM EGTA; then was similar to that of [3H]InsP3. Data are expressed as percentage Ca2+ was added to 2.2 mM. Bars indicate the time of addition of increase in 3H content of each fraction relative to 3H content (lOO1o) lipoproteins, ATP, or Ca2+. Data are shown from a representative in the respective controls at each time point. Radioactivity (dpm per experiment. Four experiments, with 60 cells from four different cell x ± ± 0.5 106 cells) at zero time was 2238 199 and 1025 114 for isolations, were done. [3H]inositol monophosphate and [3H]InsP3, respectively. The 3H content in the different inositol phosphate fractions in controls did cellular Ca2+. Both the magnitude and the time course of the not change during at least 60 min of incubation (data not shown). response among Results represent means SDs of triplicate plates from three of [Ca2+] varied cells. experiments, each with a different preparation of type II cells. Effect of apoE and apoA-I on Surfactant Secretion, Inositol Phospholipid Catabolism, and PKC Translocation. To test with cells isolated from three different preparations of type II whether the effects of LDL and HDL were specific, we used cells, mean basal [Ca2+]j was 100 ± 12 nM (n = 32 cells). ATP several different experimental approaches. (i) We examined (1 ,.M) caused an increase to 626 ± 75 nM (n = 6 cells); LDL whether the stimulatory effects of lipoproteins were due to caused an increase to 372 ± 22 nM (n = 10 cells), and HDL nonspecific effects, such as alterations in the lipid composi- caused an increase to 275 ± 25 nM (n = 12 cells). In the tion of type II cell plasma membranes. We tested whether presence of extracellular Ca2+, HDL and LDL induced a unilamellar PtdCho liposomes affected surfactant secretion rapid increase in [Ca2+]i (Fig. 4 C and E). In the absence of and inositol phospholipid catabolism. PtdCho liposomes by extracellular Ca2+, the effects ofHDL and LDL were similar themselves had no effect on either secretion or inositol to that of ATP in that the initial increase in [Ca2+]i was phospholipid catabolism (Table 1). followed by a decrease to basal levels (Fig. 4 D and F). (ii) We tested whether the effect of LDL was mediated by Addition of extracellular Ca2+ after [Ca2+]i had returned to interaction ofLDL with the classical LDL receptor. Heparin, baseline caused a second increase in [Ca2+]j. These findings which prevents binding of LDL to the LDL receptor (7), are with the that compatible hypothesis lipoproteins triggered completely blocked the stimulatory effects of LDL on sur- a release of [Ca2+J1 from intracellular stores but that a factant secretion and inositol phospholipid catabolism (Table sustained increase in [Ca2i]1 results from an influx of extra- 1). (iii) We tested whether purified apolipoproteins repro- duced the effects of LDL and HDL. ApoE binds to the LDL receptor with an affinity =20-fold higher than , the major LDL apolipoprotein (29). PtdCho-apoE com- plexes reproduced the effects of LDL on phospholipid se- cretion and PKC translocation (Table 1). It has been reported that several cell types have specific binding sites for HDL and apoA-I (39). Heparin has no effect on this binding (J. Oram, HDL LDL personal communication). In contrast with its ability to block the cellular responses to LDL, heparin had little, if any, FIG. 3. Effect of LDL and HDL on PKC activity in type II cell inhibitory effect on HDL-stimulated secretion (Table 1). membranes. Cells were incubated with 1 ELM PMA for 10 min or with ApoA-I constitutes 70-75% of the protein component of 25 ,ug of LDL per ml or 25 ug of HDL per ml for 5 min. Cell se- membranes were isolated, and PKC activity was determined, as HDL. PtdCho-apoA-I complexes stimulated surfactant described. Results represent means + SDs of duplicate plates from cretion and activated inositol phospholipid turnover in type three experiments, each with a different preparation of type II cells. II cells (Table 1). Downloaded by guest on September 28, 2021 Cell Biology: Voyno-Yasenetskaya et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4259 Table 1. Effect of LDL, HDL, and apolipoproteins on surfactant 5). However, in type II cells and in type II cell membranes, secretion, inositol phospholipid turnover, and PKC translocation this band remained present even under reducing conditions, in type II cells whereas the 230-kDa band was not seen in hepatic tissue [3H]PtdCho [3H]InsP PKC activity, under reducing conditions (data not shown). These findings secretion, % formation, % % of control were reproduced in three different preparations of type II Agent of control (n) of control (n) (n) cells and in one preparation of membranes prepared from type II cells, using four different polyclonal antisera to the ATP (0.1 mM) 603 ± 69* (6) 300 ± 34* (5) LDL receptor, one raised against the rat hepatic LDL PMA (1 ,uM) 750 ± 60* (3) 99 ± lOt (3) 400 ± 38* (5) receptor and three raised against different preparations ofthe LDL (25 ,ug/ml) 230 ± 30* (5) 172 ± 20t (3) 226 ± 29* (4) bovine adrenal LDL receptor. Heparin + LDL 95 + 10t (3) 99 ± 7t (3) 112 ± lit (3) HDL (25 ,ug/ml) 409 ± 46* (5) 224 ± 31* (4) 158 ± 16t (3) Heparin + HDL 363 ± 40 (3) DISCUSSION PtdCho liposomes The regulation of secretion of pulmonary surfactant has been (25 ,ug/ml) 99 ± 8t (3) 108 ± 7t (3) 99 ± 6t (3) studied extensively. Various secretagogues have been iden- ApoE-PtdCho tified, including hormones and vasoactive compounds, which (1.3 ,ig/ml) 225 ± 29* (3) 178 ± 1St (3) 287 ± 20* (3) activate second-messenger systems such as cAMP (6), ApoAl-PtdCho [Ca2+], (21, 22), and PKC (23). In addition, a nonhormonal (1.3 ,ug/ml) 190 ± 12 (3) 143 ± llt (3) stimulus, mechanical stretch, stimulates calcium mobiliza- ApoAl-PtdCho tion and secretion (33). In this study we have demonstrated (19 ug/ml) 283 ± 31 (3) 173 ± 18t (3) that LDL and HDL, previously unsuspected secretagogues Data are expressed as a percentage of control values. Control for type II cells, can stimulate surfactant secretion in type II value for [3H]PtdCho secretion was 1.9 ± 0.3%, n = 10. Control cells. Both apoE-PtdCho complexes and apoA-I-PtdCho value for inositol phosphate accumulation (accumulation of all frac- complexes also stimulate secretion. An increase in [Ca2+],, tions of inositol phosphates) was 2398 ± 165 dpm, n = 6. Control stimulation of inositol phospholipid metabolism, and PKC value for PKC was 50 ± 6 pmol of 32P.min-1'mg-1. Each data point translocation precede secretion, suggesting that the genera- represents, at least, triplicate plates of cells from each experiment; HDL is the number of experiments (each with a different preparation of type tion of signal-transducing molecules by LDL and II cells) is indicated in parentheses. *, P < 0.001; t, P < 0.05, responsible for the secretory response induced by those according to Student's t test. lipoproteins. The rapid generation of InsP3 in alveolar type II cells exposed to either LDL or HDL provides evidence that Presence of LDL Receptors in Type II Alveolar Cells. LDL both lipoproteins activate phospholipase C-mediated inositol receptors are present in a wide variety of cell types (7). phospholipid turnover. It seems likely that subsequent for- Although the lung has been reported to take up LDL in vivo mation of diacylglycerol and PKC activation are induced, (41) and to take up LDL and HDL in perfused lungs (5), it was followed by surfactant secretion. not known whether type II cells themselves expressed the Stimulation of [3H]PtdCho secretion, inositol phospholipid classical LDL receptor. By immunoblotting with specific catabolism, and PKC translocation by LDL in type II cells to antisera to the LDL receptor, we found that membranes occurs at concentrations of LDL comparable its reported affinity for the classical LDL receptor (7). Heparin, which prepared from freshly isolated type II cells and from type II inhibits LDL binding to its receptor, (7) completely pre- cells cultured for 22 hr express LDL receptors (Fig. 5). Rat vented the LDL-mediated stimulation of secretion of[3H]Ptd- type II cell membranes separated by SDS/PAGE under both Cho and inositol phospholipid catabolism, suggesting that reducing and nonreducing conditions showed two major specific ligand-receptor interaction is required for the LDL- bands, one that migrated in parallel with the rat LDL induced effects. It seems likely that LDL and HDL do not receptor at -130 kDa and one that migrated faster at =120 merely act by inducing nonspecific events within the plasma kDa (Fig. 5). A third band at -230 kDa also was seen. Under membrane because unilamellar PtdCho liposomes did not nonreducing conditions, the band at 230 kDa migrated with affect either the secretion of [3H]PtdCho or inositol phos- an Rf similar to that of the rat liver LDL receptor dimer (Fig. phate formation. Furthermore, apoE-PtdCho complexes, which presumably bind to the LDL receptor, stimulated 1 2 3 4 secretion of [3H]PtdCho and PKC translocation in alveolar type II cells. Type II cells, both freshly isolated and in culture, contained immunologically recognizable LDL recep- 205 5 tors (Fig. 5). Taken together, these data suggest that LDL- mediated PtdCho secretion, activation of inositol phospho- lipid catabolism, and PKC translocation are induced by LDL interaction with its receptor. 116- HDL interacts with a number of tissues and cell types to alter cellular cholesterol metabolism (8-11, 39). Our results showing that HDL and its major apolipoprotein, apoA-I, stimulate signal transduction and surfactant secretion suggest that HDL elicits these events via an apoA-I recognition site similar to that described in other cells (39). The LDL- and HDL-mediated activation of inositol phos- pholipid catabolism may stimulate secretion in type II cells FIG. 5. LDL-receptor content in type II cell membranes before via effects on cytoskeletal elements. Actin is believed to play and after 22-hr culture, as determined by immunoblotting. Fifty a role in surfactant secretion (42). The polymerization ofactin micrograms of membrane protein was run in lanes 1-3; five micro- at de novo nucleation sites on the inner leaflet of the plasma grams of membrane protein was run in lane 4. Lanes: 1, membranes membrane is stimulated by diacylglycerol generated during from freshly isolated type II cells; 2, membranes from type II cells cultured for 22 hr; 3, rat liver membranes; 4, rat liver membranes inositol phospholipid hydrolysis (43). Cytoskeletal rearrange- from ethynylestradiol-treated rats. An antibody raised against rat ments may also be involved in initiation of receptor-mediated hepatic LDL receptor was used in this immunoblot. endocytosis (44). Downloaded by guest on September 28, 2021 4260 Cell Biology: Voyno-Yasenetskaya et al. Proc. Natl. Acad. Sci. USA 90 (1993) The function of the LDL receptor or HDL-binding sites in 10. Slotte, J. P., Oram, J. F. & Bierman, E. L. (1987) J. Biol. the alveolar epithelial type II cell is unknown. We hypothe- Chem. 262, 12904-12907. size that LDL receptors participate in the delivery of lipids, 11. Oram, J. F., Johnson, C. J. & Brown, T. A. (1987) J. Biol. particularly cholesterol, which are necessary for surfactant Chem. 262, 2405-2410. synthesis. It has been suggested (3, 5) that much of lung 12. Tauber, J. P., Cheng, J. & Gospodarowicz, D. (1980) J. Clin. surfactant cholesterol is derived from serum lipoproteins Invest. 66, 698-708. 13. Fleisher, L. N., Tall, A. R., Witte, L. D., Miller, R. W. & rather than by de novo synthesis. In the isolated perfused rat Cannon, P. J. (1982) J. Biol. Chem. 257, 6653-6655. lung, Hass and Longmore (3, 5) showed that both serum LDL 14. Pomerantz, K. B., Tall, A. R., Feinmark, S. J. & Cannon, P. J. and HDL delivered radiolabeled cholesterol first to lamellar (1984) Circ. Res. 54, 554-565. bodies and subsequently to lavaged surfactant. These authors 15. Wosu, L., Parisella, R. & Kalant, N. (1983) 48, hypothesized that LDL was taken up as an intact particle, 205-220. whereas HDL transferred cholesterol without mandatory 16. Block, L. H., Knorr, M., Voot, E., Locher, R., Vetter, W., uptake of the protein moiety, consistent with the subse- Groscurth, P., Qiao, B.-Y., Pometta, D., James, R., Regenass, quently described selective-uptake pathway (9). It is possible M. & Pletscher, A. (1988) Proc. Natl. Acad. Sci. USA 85, that the stimulatory effects of both HDL and LDL on 885-889. surfactant secretion in type II cells contribute to a basal level 17. Bockov, V. N., Voyno-Yasenetskaya, T. A. & Tkachuk, of secretion, which then is further modulated by hormones V. A. (1991) Biochim. Biophys. Acta 1097, 123-127. and vasoactive agents. However, hormones and vasoactive 18. Smirnov, V. N., Voyno-Yasenetskaya, T. A., Antonov, A. S., Lukashev, M. E., Shirinsky, V. P., Tertov, V. V. & Tkachuk, agents also may alter surfactant secretion by altering LDL V. A. (1990) Annu. N. Y. Acad. Sci. 598, 167-181. receptors or HDL-binding sites in type II cells. 19. Scott-Burden, T., Resnick, T. J., Hahn, A. W. A., Baur, U., LDL receptors may have specialized roles in type II cells. Box, R. J. & Buhler, F. R. (1989) J. Biol. Chem. 264, 12582- The observations that type II cells contain more than one 12589. monomeric form of the LDL receptor (120 kDa and 130 kDa) 20. Voyno-Yasenetskaya, T. A., Dobbs, L. G. & Williams, M. C. and that the 230-kDa form ofthe LDL receptor of type II cells (1991) Am. J. Physiol. Suppl. 261, 105-109. is present under reducing conditions, unlike the 230-kDa 21. Sano, K., Voelker, D. R. & Mason, R. J. (1987)Am. J. Physiol. band of the hepatic or fibroblast LDL receptor, suggest that 253, C679-C686. these receptors in type II cells may have some specialized 22. Pian, M. S., Dobbs, L. G. & Duzgunes, N. (1988) Biochim. functions or intracellular itineraries. In renal tubular epithe- Biophys. Acta 960, 43-53. lial cells, LDL receptors located on the basal side of the cell 23. Sano, K., Voelker, D. R. & Mason, R. J. (1985) J. Biol. Chem. (which is in contact with plasma) and LDL receptors located 260, 12725-12729. on the apical side of the cell (which is in contact with the 24. Ruoslahti, E., Hayman, E. G., Pierbacher, M. & Engvall, E. (1982) Methods Enzymol. 82, 803-831. tubular lumen) differ functionally (45). LDL receptors lo- 25. Dobbs, L. G., Gonzalez, R. & Williams, M. C. (1986)Am. Rev. cated on the basal side of the cell participate in the control of Respir. Dis. 131, 141-145. cell cholesterol homeostasis, whereas LDL receptors on the 26. Havel, R. J., Eder, H. & Bragdon, H. F. (1955) J. Clin. Invest. apical side appear to be responsible for the transport of LDL 34, 1345-1353. to the basal side (45). 27. Lowry, 0. H., Rosenbrough, M. G., Farr, A. L. & Randall, Investigation of interactions between lipoprotein-receptor R. H. (1951) J. Biol. Chem. 193, 265-275. endocytosis, cytoskeletal rearrangement, and surfactant se- 28. Hamilton, R. L., Goerke, J., Guo, L., Williams, M. C. & cretion may facilitate elucidation of the mechanisms of how Havel, R. J. (1980) J. Lipid Res. 21, 981-992. activation of second-messenger systems participates in the 29. Pitas, R. E., Innerarity, T. L. & Mahley, R. W. (1980) J. Biol. metabolism of HDL and LDL. Further, the effects of lipo- Chem. 255, 5454-5460. proteins on type II cells may 30. Dobbs, L. G., Gonzalez, R. F., Marinari, L. A., Mescher, represent a series of membrane E. J. & Hawgood, S. (1986) Biochim. Biophys. Acta 877, trafficking events that couple and coordinate specific en- 305-313. docytic and exocytic processes. 31. Berridge, M. J., Dawson, R. M. C., Downes, C. P., Heslop, J. P. & Irvine, R. F. (1983) Biochem. J. 212, 473-482. We thank Dr. Richard Havel (Cardiovascular Research Institute, 32. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S. & Nish- University of California at San Francisco) and Dr. Janet Boyles izuka, Y. (1982) J. Biol. Chem. 257, 13341-13348. (Gladstone Foundation Laboratories, University of California at San 33. Wirtz, H. R. W. & Dobbs, L. G. (1990) Science 250, 1266- Francisco) for their gifts of LDL-receptor-specific antisera, Dr. 1269. Phoebe Fielding (Cardiovascular Research Institute, University of 34. Grynkewitz, G., Poenic, M. & Tsien, R. Y. (1985) J. Biol. California at San Francisco) for the gift of HDL and LDL and for Chem. 260, 3440-3450. helpful discussions, Mr. Steven Lear (Veterans Administration Med- 35. Wade, M. H. & McQuiston, S. A. (1988) Training Manual ical Center) for technical assistance with the immunoblotting, and (Meridan Instruments, Okemos, MI), p. 37. Edward Hamilton for manuscript preparation. This work was sup- 36. Erickson, S. K., Lear, S. R., Barker, M. E. & Musliner, T. A. ported, in part, by grants from the National Heart, Lung, and Blood (1990) J. Lipid Res. 31, 933-945. Institute HL41958 (T.A.V.-Y., L.G.D.), 24075 (R.L.H., L.G.D.), 37. Rice, W. R. & Singleton, F. M. (1986) Br. J. Pharmacol. 89, and the Department of Veterans Affairs (S.K.E.). 485-491. 38. Gilfillan, A. M. & Rooney, S. A. (1987) Biochim. Biophys. 1. King, R. J. & Clements, J. A. (1972) Am. J. Physiol. 223, Acta 917, 18-23. 715-726. 39. Mendez, A. J., Oram, J. F. & Bierman, E. L. (1991) J. Biol. 2. King, R. J. (1974) Fed. Proc. 33, 2238-2247. Chem. 266, 10104-10111. 3. Hass, M. A. & Longmore, W. J. (1979) Biochim. Biophys. Acta 40. Dorm, C. C., Rice, W. R. & Singleton, F. M. (1989) Br. J. 573, 166-174. Pharmacol. 97, 163-170. 4. Turley, S. D., Andersen, J. M. & Dietschy, J. M. (1981) J. 41. Spady, D.