Introduction

Abstract Introduction Materials and Methods Results Discussion References

Heterotrimeric guanine nucleotide-binding (G) proteins transduce a wide variety of receptor-mediated signals to effectors that are involved in numerous cell functions, including cell differentiation, growth, and apoptosis. G protein holoenzyme identity is conferred by at least 20 different  subunits, and the effector signaling is generated by either the  or the subunit, depending on the pathway and cell type involved. G proteins alter cell growth and differentiation by activating kinase cascades that converge in the nucleus to alter gene expression (1). The G protein  subunits are grouped into four subfamilies based on amino acid sequences, response to toxins, and functional properties. Pertussis toxin (PTX), which decouples a subfamily of G proteins (Gi), attenuates both Gi inhibition of adenylyl cyclase in platelets and mitogenic responses to thrombin and bombesin/gastrin-releasing peptide (2). Both effects of PTX can be mimicked by antibodies to one particular family member, Gi2 (3). Furthermore, cell growth is specifically linked to Gi2 via mitogen-activated protein kinase (MAPK) in LLC-PK1 cells, a renal epithelial cell line (4). Gi2 has been implicated as a regulator of more complex biologic processes, such as differentiation and development (5, 6). Specifically, Gi2 has been implicated in the induction by retinoic acid of F9 teratocarcinoma cells to become primitive endoderm (6). Also, transgenic mice that express an antisense construct to Gi2 in the liver and adipose tissue show impaired growth of these organs (7, 8).

There is a paucity of data on the role of G proteins in lung growth and development. A role for G proteins in these processes is likely, because a number of growth receptors that are critical to lung development, including thrombin, bombesin/gastrin-releasing peptide, and vasopressin, are coupled to PTX-sensitive G proteins (2). Gastrin-releasing peptide, and possibly other bombesin-like peptides that mediate their signals via G protein-coupled receptors, are secreted by pulmonary neuroendocrine cells (9, 10). A functional role for these peptides is supported by their ability to stimulate lung cell proliferation and type II cell differentiation both in vitro and in vivo during the late stages of lung development. Bombesin-like peptides mediate their signals via G protein-coupled receptors, particularly after embryonic Day 18 (E18) in rats. Also, epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), which are critical to lung development, interact with G protein signaling pathways (11, 12).

In view of the abundant evidence for the presence of the G proteins in the signaling pathways that are critical to lung development, we embarked on studies to establish the presence of a key member of the Gi subgroup, Gi2, in developing lung epithelium, and attempted to correlate the time course and location of its expression with a role in growth. The present study shows that Gi2 is expressed throughout rat fetal lung development, with increased expression during late fetal and early postnatal development. Expression of Gi2 is enhanced with the growth of fetal airway epithelium. There was a strong correlation of Gi2 expression with activation of MAPKs. Gi2 is predominantly localized on the cell surface membrane. A role for Gi2 in growth is likely because DNA synthesis in fetal airway cells was inhibited by PTX.

	Materials and Methods

Abstract Introduction Materials and Methods Results Discussion References

Cell Culture

Fetal airway epithelial cells were obtained from Sprague- Dawley rats (13, 14). Three pathogen-free, pregnant Sprague-Dawley rats were used per experiment. Rats of Day 19 gestation were anesthetized with an intraperitoneal injection of pentobarbital sodium (65 mg/100 g). The uterus was removed. The fetuses were decapitated in utero and subsequently removed from their dams. Lungs were removed aseptically and dissected free of trachea, esophagus, thymus, and heart. The whole lungs were minced and incubated in 1 µg/ml collagenase A (Boehringer Mannheim, Chicago, IL) in Hanks' balanced salt solution (HBSS; GIBCO, Rockville, MD) for 20 min at 37°C (approximately 30 ml per liter). Large tissue fragments were removed by filtering through sterile gauze. The mixed cell suspension was precipitated by centrifugation at 120 × g for 4 min. The cells were washed in HBSS and then resuspended in 50 ml of Dulbecco's modified Eagle's medium (DMEM). Cells were then plated on plastic for 20 min to remove the mesenchymal cells. The supernatant was transferred to immunoglobulin G (IgG)-coated petri dishes and incubated at 37°C for 60 min to enrich for type II pneumocytes (which are nonadherent to IgG) (14, 15). The supernatant was poured onto 10-cm tissue culture plates and the cells were grown for 4 d. The medium was changed after 24 h.

Media

Primary cells were grown as monolayers and maintained in DMEM containing 10% fetal calf serum (FCS) in a 5% CO₂ atmosphere.

Immunoblotting of Gi2

Cells were washed twice in phosphate-buffered saline (PBS; without calcium or magnesium), and were then lysed by addition of 0.3 ml of lysis buffer (20 mM 4-[2-hydroxyethyl]-1-piperazine-N'-2-ethanesulfonic acid [Hepes], pH 7.4; 2 mM ethyleneglycol-bis-[-aminoethyl ether]-N,N'-tetraacetic acid [EGTA]; 50 mM -glycerophosphate; 1 mM dithiothreitol [DTT]; 1 mM Na₃VO₄; 1% Triton X-100; 10% glycerol; and 1× Complete protease inhibitor cocktail [Boehringer Mannheim]). Scraped lysates were solubilized by boiling in Laemmli's buffer (100 mM Tris, pH 6.8; 2% sodium dodecyl sulfate [SDS]; 2% 2-mercaptoethanol; 10% glycerol; and 0.01% bromphenol blue), and were loaded onto a 12% acrylamide gel, with 50 µg of protein loaded per lane. Following SDS-polyacrylamide gel electrophoresis, proteins were transferred onto an Immobilon membrane (Millipore, Bedford, MA), and the membrane was then stained with Coomassie blue to ensure that all lanes contained equivalent amounts of transferred protein. The destained membrane was then blocked in blotting buffer (5% nonfat dry milk in 20 mM Tris, pH 7.4, with 0.15 M NaCl and 0.1% Tween 20), and was incubated with antibodies. Antibody-bound proteins were reacted with an enhanced chemiluminescent detection system as described by the manufacturer (Amersham, Arlington Heights, IL), followed by autoradiography.

Antibodies

The two antibodies used to detect Gi2 are AS7 and Gi2-4E. The former is a rabbit polyclonal antibody directed against amino acids 345-354 of Gi2, and is available from NEN (Boston, MA) (16). It has been widely used and reacts with Gi2 and Gi1. Gi1 is not expressed in lung (17). Gi2-4E is a rabbit polyclonal antibody that was generated in Massachusetts General Hospital (Boston, MA) against amino acids 100-156 of Gi2, and has strong specificity for Gi2 (T. B. Kinane and L. Ercolani, manuscript in preparation). Initially, all experiments were performed with Gi2-4E and were confirmed with AS7.

Immunofluorescence

Fetal cells grown on glass coverslips were fixed in 4% paraformaldehyde. Cells were permeabilized with 0.1% Triton X-100 and blocked with 1% bovine serum albumin for 1 h. The coverslips were incubated for 1 h with anti-Gi2 antibody (dilution = 1:100 for both Gi2-4E and AS7). The coverslips were washed three times with PBS and were then incubated with Cy3-conjugated antirabbit antibody (dilution = 1:800).

Protein Assay

Protein assay was done with the dye-binding assay of Bradford as described by the manufacturer (Bio-Rad, Hercules, CA).

Kinase Assays

Immunoprecipitation of MAPK. Cells were washed twice with ice-cold PBS and lysed in lysis buffer (20 mM Hepes, pH 7.4; 2 mM EGTA; 50 mM -glycerophosphate; 1 mM DTT; 1 mM Na₃VO₄; 1% Triton X-100; 10% glycerol; 2 µM leupeptin; 10 kallekrein-inhibiting units per ml of Traysylol; and 400 µM phenylmethylsulfonyl fluoride). Insoluble material was removed by centrifugation at 8,000 × g for 10 min at 4°C. The supernatants were matched for 400 µg of protein content. To the supernatants were added 3 µl of polyclonal antibody recognizing p42 MAPK (amino acids 305-327; Santa Cruz Biotechnology, Santa Cruz, CA), 3 µl of polyclonal antibody recognizing p44 MAPK (amino acids 345-358; Santa Cruz Biotechnology), and 40 µl of 50% protein A Sepharose beads. This mixture was incubated for 3 h.

MAPK assay. For measurement of MAPK activity, the immunocomplexes were collected by centrifugation and washed three times with lysis buffer, three times with lithium chloride wash solution (500 mM LiCl; 100 mM Tris-HCl, pH 7.6; 0.1% Triton X-100; 1 mM DTT), and three times in assay buffer (20 mM 3-[N-morpholino]propanesulfonic acid, pH 7.2; 2 mM EGTA; 10 mM MgCl; and 1 mM DTT). To 40 µl of 50% protein A-Sepharose beads was added 20 µl of myelin basic protein (MBP) (Sigma Chemical Co., St. Louis, MO) diluted to 5 mg/ml. To this mix was added 15 µl of reaction mixture (50 mM MgCl₂, 0.5 mM [-³²P]adenosine triphosphate 2-10,000 CPM/pmol]). The reaction was initiated by incubating the mixture for 20 min at 30°C. The reaction was terminated by adding 25 µl of 6× Laemmli's buffer and boiling for 5 min. The samples were run on 15% low-bis SDS gel. Gels were stained with Coomassie brilliant blue, and kinase activity was measured by cutting out the MBP bands and measuring their radioactivity in a liquid scintillation counter.

Cell Proliferation Studies

DNA synthesis was monitored by 5-bromo-2'-deoxyuridine (BrdU) labeling of the DNA of mitotically active cells. Studies were done with an enzyme-linked immunosorbent assay (ELISA) system from Boehringer Mannheim (18) in accordance with the manufacturer's guidelines. The following conditions were used: fetal airway cells were cultured in a 96-well plate with 5,000 cells per well. Cells were grown for 48 h, and PTX or vehicle was added for 12 h. Subsequently, BrdU was added to the cells and the cells were reincubated for 16 h. After removal of the culture medium, the cells were fixed and their DNA was denatured in one step by adding FixDenat (Boehringer Mannheim). Anti-BrdU working solution (a solution with a peroxidase-conjugated monoclonal antibody against BrdU) was added for 90 min at room temperature. Antibody was removed and wells were rinsed three times with 200 µl/well of washing solution. An aliquot of 25 µl 1 M H₂SO₄ was added to each well, and the absorbance was measured at 450 nm.

	Results

Abstract Introduction Materials and Methods Results Discussion References

Expression of Gi2 during Lung Development

To define the expression of Gi2 throughout lung development, lungs were obtained from fetal Sprague-Dawley rats from Days 16 through 21 and on postnatal Days 1 and 2. Extracts of these lungs were immunoblotted with anti-Gi2 antibody (Gi2-4E) (Figure 1). Gi2 protein was expressed throughout lung development, but showed increased expression during fetal Days 19 through 21 and during the early postnatal period. This augmentation of expression of Gi2 occurs during a time when alveolar development is beginning. These immunoblots showed no nonspecific bands. Similar results were obtained from three immunoblots. The combined densitometric scan data for each immunoblot confirmed this profile.

View larger version (38K): [in this window] [in a new window]   Figure 1.   Gi2 expression in lung development. Lungs were obtained from Sprague-Dawley rats during embryonic Days 16 to 21 and postnatal Days 1 and 2. Lungs were homogenized. Lungs from 10 fetuses or newborns from five litters were pooled, and immunoblotting was performed with an aliquot from this pool. The lower panel is a representative immunoblot of total extract from each day performed with rabbit IgG Gi2-4E. The experiment was repeated three times. The profile in all three experiments was similar. The upper panel shows the combined densitometric scan value of each immunoblotting, and the error bars reflect the SEM. Because each immunoblot was performed on a different day, Day 18, on which there was significantly less expression of Gi2 than on other days in each experiment, was assigned the value of 100 densitometric units.

Gi2 Is Present in the Developing Lung Epithelium on the Plasma Membrane and in the Golgi Apparatus

The lung develops as a ventral outpouching of the primitive gut at about Day 12.5 of gestation in the rat. The primary bronchus elongates into the mesenchyme and branches into the two main brochi. The lung continues to develop into early postnatal life. To define the cellular and subcellular location of Gi2 during lung development, lungs were obtained from postnatal Day 1 Sprague-Dawley rats, and were fixed in 4% paraformaldehyde and sectioned. Immunofluorescence was used to localize Gi2 with a polyclonal antibody against Gi2 (Gi2-4E) as the primary antibody (dilution = 1:100) and a Cy3-conjugated antirabbit antibody (dilution = 1:800) as the secondary antibody (Figure 2A). In postnatal lungs the linear staining was consistent with membrane staining, and the perinuclear staining was consistent with Golgi staining (Figure 2A). Almost identical staining was seen in lungs obtained at embryonic Day 19 (data not shown).

View larger version (78K): [in this window] [in a new window]   Figure 2.   Immunofluorescent localization of Gi2 in the lung of postnatal Day 1 rat. (A) Sections of lungs from postnatal Day 1 (magnification: ×120) were fixed, permeabilized, and stained to produce immunofluorescence with immune rabbit IgG Gi2-4E. Linear staining (white arrow) in the epithelium around the alveolus (A) on postnatal Day 1 suggested membrane staining. Non-plasma-membrane staining is also seen (yellow arrow). (B) Colocalization studies were performed on postnatal Day 1 sections. Sections from postnatal Day 1 were stained to produce immunofluorescence with both immune rabbit IgG Gi2-4E and the monoclonal antibody P200 (a Golgi marker). P200 was detected as green because the secondary antibody was FITC-conjugated; Gi2 was detected as red because the secondary antibody was Cy3-conjugated and the overlap was yellow. There was some Golgi staining (yellow arrow). Membrane staining is also seen (red arrow).

To define clearly both the cellular and subcellular locations of Gi2, colocalization studies were performed with P200, a Golgi marker (Figure 2B) (monoclonal antibodies to P200 were provided by D. Brown, Ph.D., of the Massachusetts General Hospital) (19), and Gi2. P200 was detected as green because the secondary antibody was fluorescein isothiocyanate (FITC)-conjugated, Gi2 was detected as red because the secondary antibody was Cy3- conjugated, and the overlap was yellow. There was some overlap consistent with Golgi staining for Gi2. However, there was widespread expression of Gi2 outside the Golgi apparatus as well.

Gi2 Is Present on the Plasma Membrane and in the Golgi Apparatus in Fetal Airway Cells Grown in Primary Culture

The expression of Gi2 in developing lung was further explored in primary culture of fetal airway cells. Developing epithelium was derived from E19 Sprague-Dawley rats and was grown in primary culture in the presence of antibiotics. Cells growing on glass coverslips were fixed. There was bright membrane staining as well as Golgi staining with Gi2-4E (Figure 3). Gi2 in both of these locations was confirmed with the second antibody to Gi2 (AS7; NEN). In other secretory epithelial cells, another PTX-sensitive G protein, Gi3, is localized to the Golgi apparatus and regulates transport in it. Neither of the two antibodies (Gi2-4E or ASF7) detect Gi3, and lack of specificity of the antibodies is unlikely to be an explanation for the Golgi staining. This may suggest an additional role for Gi2 in Golgi trafficking, and Gi2 may take the role of Gi3 in the regulation of secretion in fetal airway cells.

View larger version (34K): [in this window] [in a new window]   Figure 3.   Immunofluorescence in primary cultures of fetal airway cells confirms that Gi2 is present, primarily on the plasma membrane and in the Golgi apparatus. Primary cultures of fetal airway cells grown for 3 d were fixed, permeabilized, and stained to produce immunofluorescence with immune IgG Gi2-4E. Representative sections show strong membrane staining (white arrow) and Golgi staining (yellow arrow). Bottom frame shows membrane staining of the area delineated by the white box at a higher magnification.

Growth of Primary Pulmonary Epithelial Cells Is Inhibited by PTX

The expression of Gi2 in developing lung is consistent with its known growth-regulatory role in other cell types. Because Gi2 belongs to the Gi family, growth of pulmonary epithelial cells should be PTX sensitive if Gi2 is linked to growth pathways. PTX ribosylates the Gi family of G proteins and prevents dissociation of the Gi protien heterotrimer into  and subunits. To define the effect of Gi in fetal pulmonary epithelial cells, PTX was added to E19 fetal airway cells grown in 5% FCS to final concentrations of 0.1, 1, and 10 ng per milliliter of medium. There was a dose-dependent inhibition of DNA synthesis as measured by BrdU incorporation in primary cultures of these cells (Figure 4). The BrdU incorporation was measured with an ELISA. Thus, PTX-sensitive G proteins, of which Gi2 is a key member, are likely to play a significant role in the growth of developing pulmonary epithelial cells.

View larger version (30K): [in this window] [in a new window]   Figure 4.   Proliferation of fetal airway cells is inhibited by PTX. Fetal airway cells were cultured in a 96-well plate with 5,000 cells per well. Cells were grown for 48 h, and PTX (0.1 ng/ml, 1 ng/ml, and 10 ng/ml) was added for 12 h. Subsequently, BrdU was added to the cells and the cells were reincubated for 16 h. BrdU incorporation was measured with an ELISA. The experiment was repeated three times. The error bars reflect the SEM.

Gi2 Expression Is Increased with Growth

The expression of a number of key regulators of mitogenesis increases with growth. To define the expression of Gi2 during the growth of airway epithelium, primary cultures of fetal epithelial cells were derived from E19 Sprague- Dawley rats. Cell cultures were limited to 4 d in duration because after that time the type II cells undergo dedifferentiation. At Day 2, cells were approximately 20% confluent; at Day 3, 30% confluent; and at Day 4, 60% confluent. Immunoblotting of extracts was done with antibody against Gi2 (Gi2-4E) (Figure 5). Gi2 was readily detected, and its expression increased with time. Thus, its expression parallels the growth of fetal epithelial cells, which were actively dividing at 60% confluence. Growth should decrease with further culture. However, extended culture was not done because of the risk of dedifferentiation. The increased expression of Gi2 with cell growth is consistent with its involvement in growth pathways. This growth profile is not found in fibroblasts (data not shown). It is unlikely that this increase in Gi2 expression is linked to dedifferentiation of fetal airway epithelial cells, because dedifferentiation is not apparent until Day 6 of culture.

View larger version (57K): [in this window] [in a new window]   Figure 5.   Gi2 has increased expression with growth of fetal airway cells. Immunoblot of extracts of primary cultures of fetal airway cells (top panel ). Primary cultures of fetal airway cells were harvested at approximately 20%, 30%, and 50% of confluence. These confluences occurred at Days 2, 3, and 5, respectively. The gel was stained with Coomassie blue to confirm equal loading (middle panel ). MAPK is a critical modulator of growth and is a marker of growth. With the same extracts used for immunoblotting to determine Gi2 expression, the relative activation of MAPK44 was determined by immunoprecipitating MAPK42 and MAPK44 and determining their ability to phosphorylate MBP (bottom panel ).

Mitogenic pathways are linked to downstream intracellular phosphorylation cascades that can be initiated by growth factor-type receptors that bind such ligands as insulin, EGF, and PDGF and also by G protein-coupled receptors. The MAPKs (also called extracellular signal-regulated kinases) are important regulatory kinases in these cascades, integrating and amplifying signals from a number of different growth factors. Using the same extracts with which we did immunoblotting for Gi2, we determined the relative activation of MAPK44 by immunoprecipitating MAPK and assaying its ability to phosphorylate MBP. With increasing Gi2 expression there was increasing MAPK44 activation (Figure 4). Thus, the timing of Gi2 expression strongly correlates with the activation of MAPK, a known marker of cell growth.

	Discussion

Abstract Introduction Materials and Methods Results Discussion References

G proteins are critical in the regulation of mitogenesis. PTX inhibits the mitogenic responses induced by growth factors known to be involved in lung development, including thrombin and bombesin/gastrin-releasing peptide (2). These responses are especially seen in cells in which Gi2 and Gi3 are predominantly expressed. In addition to a role in mitogenesis, a role for G proteins in the regulation of development has been established. In Dictyostelium, differentiation is triggered by a cyclic adenosine monophosphate (cAMP)-dependent increase in the transcription of  subunit genes. Such a response is also seen in LLC-PK₁ cells, a renal epithelial cell line in which an increase in intracellular cAMP concentrations raises Gi2 protein levels 3-fold (20). Also in Dictyostelium, G4 null mutants created by gene disruption have aberrant morphologic differentiation, reduced levels of prespore gene expression, and an inability to produce spores (21). In Drosophila, gastrulation is impaired in homozygotes with mutations of the concertina gene, which encodes a G protein (22, 23). Because the gene family encoding G subunits is highly conserved in all eukaryotes, it is likely that mechanisms for their regulation of morphogenesis in higher eukaryotes have also been conserved. Gi2 has been shown to have a role in endoderm development (6) and in differentiation of kidney epithelial cells. Thus, it is likely that Gi2 regulates lung epithelial cell development.

A role for Gi2 in growth pathways is likely, but these pathways are probably redundant. In transgenic mice that express an antisense construct to Gi2 in the liver and adipose tissue, there is a marked reduction of the growth of these organs (7, 8). Yet in the Gi2-knockout mouse, organ size was normal and the mouse had an immune defect (24). In the lungs, this aberration in phenotype is partly explained by compensation by other Gi isoforms. Indeed, the investigators who made these findings with the Gi2 knockout mouse also showed that there was an upregulation of other G proteins in this mouse model (25).

Lung growth and development is coordinated by transcription factors including thyroid transcription factor (TTF)1, hepatocyte nuclear factor (HNF)-3, and N-myc (26). Knockout mice for these transcription factors have severe defects in lung development. These transcription factors regulate the expression of key lung proteins, including the surfactant-associated proteins (27). The human Gi2 promoter contains two potential binding sites for TTF1 and a partial site for HNF-3 (Figure 6) (28). This would help explain the expression of Gi2 throughout lung development. The increased expression of Gi2 during later development suggests that Gi2 is regulated by transcription factors other than TTF1, HNF-3, and N-myc. The expression of Gi2 is accentuated during later development and coincides with alveolar development. Retinoic acid is critical to this stage of lung development, and indeed, alveolar destruction by emphysema may be reversed by treatment with retinoic acid (32). In F9 teratocarcinoma cells, Gi2 participates in a pathway linked to retinoic acid-induced differentiation (6). Hence the regulated expression of Gi2 during alveolar development is consistent with its having a role in such pathways.

View larger version (14K): [in this window] [in a new window]   Figure 6.   The human Gi2 promoter contains two potential binding sites for TTF1 and a partial site for HNF-3.

Gastrin-releasing peptide, a homologue of the amphibian peptide bombesin, is present in pulmonary neuroendocrine cells and appears to be a growth factor for both normal and neoplastic pulmonary cells. Receptors for this peptide are coupled to PTX-sensitive G proteins. Of PTX-sensitive G proteins, Gi2 is the best candidate, because Gi1 and Go are not expressed in the lung (17) and Gi3 does not effect growth signals (3). The receptors for bombesin-like peptides are expressed throughout fetal lung development, with a peak in later gestation (9, 33). The correlation of Gi2 expression with the expression of bombesin-like peptide receptors is consistent with a role for Gi2 in the growth pathways involving these receptors.

Localization of protein can provide critical information about potential function. Although there is some recycling of growth receptors to intracellular vesicles, most of these receptors are localized to the plasma membrane. The localization of Gi2 to the plasma membrane of pulmonary cells is not surprising. However, a role for Gi2 other than the regulation of growth is possible. G proteins regulate vectorial ion flow across the alveolar epithelium. This flow provides the driving force for lung fluid secretion in the prenatal lung, and for fluid reabsorption at birth and thereafter into adult life (34). The direction of fluid flow is reversed at birth to bring about reabsorption of the lung fluid so that gas exchange can be initiated successfully in the neonate (34). This functional switch at birth involves active Na⁺ reabsorption. The switch is regulated by PTX-sensitive G proteins. However, Gi2 is expressed before this switch. Thus, a role for Gi2 other than the regulation of vectorial ion flow is likely.

In addition to its plasma membrane location, there is expression of Gi2 on the Golgi apparatus and diffusely throughout the cell. Takahashi demonstrated that the  subunit of Gi2 translocates from the membrane to the cytosol upon activation in mouse mastocytoma P-815 cells (35). Accordingly, the plasma-membrane staining for Gi2 may reflect the activated  subunit about to interact with an effector. Localization of Gi2 to the Golgi apparatus is unusual, but has been described in nasal epithelial cells (36). In LLC-PK1 cells, Gi3 is localized on the cytoplasmic face of Golgi cisternae, and is distributed across the whole Golgi stack (37). Overexpression of Gi3 on Golgi membranes in transfected cells retarded the production of constitutively secreted heparan sulfate proteoglycans and resulted in the accumulation of precursors in the medial trans-Golgi (37). Gi2 may replace Gi3 in fetal airway cells. However, basal secretion of the major phospholipid is PTX-insensitive in type II pneumocytes (13). The possible function for Gi2 in the regulation of secretion remains to be elucidated.

Mitogenic pathways are linked to downstream intracellular phosphorylation cascades, including MAPK cascades that can be initiated by growth factor-type receptors. MAPK is activated by a variety of extracellular stimuli, including agonists for G protein-coupled receptors such as thrombin and bombesin. Gi2 has been shown to play an important role in pathways that control cell differentiation and growth via activation of MAPK. In Rat-1a and LLC-PK1 cells, constitutively activated Gi2 activates MAPK (4, 38). The correlation of the expression of Gi2 with both the growth of primary cultured cells and the activation of MAPK strongly suggests a role for this G protein in the regulation of growth. It is plausible that Gi2 activates MAPK in primary lung cells, and this possibility is now being explored. The expression of Gi2 may be a consequence of loss of differentiation and increased mitosis. We believe that dedifferentiation is unlikely, since cell growth in our study was limited to 4 d. Cell morphology was observed daily, and no changes were noted and no overgrowth of fibroblasts was observed.

On the basis of our data, we conclude that PTX-sensitive G proteins are likely to play a significant role in the growth of developing pulmonary epithelial cells. Also, Gi2 is likely to be the key Gi family member that regulates growth, because of its site and time course of expression. Understanding the pathway in which Gi2 functions is likely to provide key insights into the regulation of growth and development of pulmonary epithelial cells.