Introduction

Top Abstract Introduction Methods Results Discussion References

Protein kinase (PK) C is a serine threonine-specific kinase that plays diverse roles in a number of cellular events. Our previous work has shown that PKC is involved in the regulation of tone of airway smooth muscle (ASM) (1, 2). Low concentrations of phorbol esters such as phorbol dibutyrate stimulate endogenous PKC to produce relaxation, and higher concentrations produce a contractile response (2). PKC is also implicated in cell replication (3), and evidence for this exists in ASM. Activation of PKC by phorbol esters stimulates proliferation in human ASM (HASM) (4), and specific inhibitors such as RO31-8220 produce inhibition of growth induced by platelet-derived growth factor (PDGF) (5).

Rather than a single enzyme, PKC consists of a family of 12 isoforms that fall into three categories (6) according to their substrate specificities and cofactor requirements (7), viz, conventional, or calcium- and diacylglycerol-dependent, (PKC-, -I, -II, and -); novel, or calcium-independent (PKC-, - , -, and -); and the atypical isoforms such as PKC-, -/. Recently, we (8, 9) and others (10, 11) have described the isoforms present in the ASM of a number of species, including humans (9). Isoforms from each of these three groups were represented in the species studied, although there did appear to be specie differences. The presence of multiple isoforms of PKC in these tissues suggests diverse roles. There is evidence to indicate that individual isoforms may be linked to specific functions (3, 12) and that expression may be altered in disease states (18). PKC-, an atypical isoform, has been linked specifically to cell proliferation, and indeed, we have found that upregulation of PKC- protein occurs in HASM cells stimulated to proliferate with fetal bovine serum (FBS) or PDGF (9). In the current study, we used adenoviral-mediated expression of an antisense of PKC- to investigate whether this upregulation of PKC- is the cause or a consequence of ASM cell proliferation.

	Methods

Top Abstract Introduction Methods Results Discussion References

Cell Isolation and Culture

HASM cells were cultured by the method of Johnson and colleagues (19). Human lung was obtained from eight patients. Four of these were undergoing transplantation: one for cystic fibrosis, one for lymphangiomyomatosis, and two for emphysema. Two patients were undergoing lung resection for malignant pulmonary lesions. One patient was a donor whose lungs had been subjected to trauma and were thus unsuitable for transplantation, and one was undergoing diagnostic biopsy. Explants of ASM were dissected from large bronchial airways (5 to 15 mm diameter) or biopsies and incubated in Dulbecco's modified Eagle's medium supplemented with 1% penicillin/streptomycin/fungizone (Life Technologies, Mulgrave, Australia) and 10% FBS (Life Technologies). Flasks were kept in incubators at 37°C in humidified air containing 5% CO₂. Smooth muscle cells were grown to confluence from the explants and used for experiments between passages 4 and 7. Cultures were confirmed as pure smooth muscle cells by fluorescent staining with monoclonal antibody specific to -smooth-muscle actin and calponin (Sigma, St. Louis, MO).

Preparation of Recombinant Adenovirus

PKC- complementary DNA (EMBL Ac. No. M94632; a kind gift from H. Mischak, Laboratory of Genetics, National Cancer Institute, Bethesda, MD) (20) was subcloned from pUC19 (by a HindIII/XbaI digest which was then blunt-ended) into the EcoRV site of pXCMV. pXCMV is an adenoviral shuttle plasmid generated by subcloning the NruI/DraIII digested and blunted expression cassette from pRcCMV (Invitrogen, Carlsbad, CA) into XbaI digested and blunted pXCX3 (pXCX3 was derived from pXCX2 [21]). The wild-type and antisense full-length double-stranded DNA clones generated were screened for orientation by restriction digest mapping and the appropriate wild-type and antisense clones were isolated. Recombinant adenovirus was prepared essentially as described by Graham and Prevec (22). Cesium chloride-purified plasmid containing the pXCMV PKC- antisense gene cassette was cotransfected with pJM17 in a ratio of 1:1 using calcium phosphate transfection. Control virus MX17 was constructed by recombination between pXCX2 and pJM17; it does not contain a gene cassette. Transfection was carried out only in low-passage (< 50) HEK 293 cells and once transfected, the HEK cells were maintained in 0.5% agarose and 1× culture medium. Recombinant viruses were isolated 1 to 2 wk later as single plaques and amplified by reinfecting confluent monolayers of HEK 293 cells. Recombinant viruses were characterized by preparing lysates of cells that showed signs of cytopathic effect 3 to 7 d after the second round of infection. Proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane for immunoblotting with anti-PKC- antibody (Santa Cruz Biotechnology, Santa Cruz, CA) to identify suitable recombinant viral clones with reduced expression of PKC-. Medium from the 35-mm dish was used in further rounds of amplification to generate stock virus, which was purified by cesium chloride gradient. Plaque assays were performed in HEK 293 cells to determine the titer (plaque-forming units [pfu]/mL) of these stocks.

Measurement of Efficiency and Toxicity of Adenoviral Infection

We used an adenovirus expressing a humanized mutant of green fluorescent protein (GFP), Ad-EF1-GFP20 (23), or CMV-GFP20 to examine the efficiency of adenoviral infection of HASM cells. In these experiments, cells grown on coverslips were infected with 0 or 100 pfu/cell Ad-EF1-GFP20 or CMV-GFP20 and quiesced with 1% FBS for 24 h. We have previously shown that after 24 h in 1% FBS, > 80% of the cells are in G₀/G₁ (24). Cells were grown in 10% FBS for 48 h and then examined by fluorescence microscopy to ascertain that expression of GFP was uniform. To examine whether infection with adenovirus affected normal proliferation of cells, cells were grown, infected, and quiesced as described earlier, although in six-well culture plates. Cells were then grown in 1, 5, and 10% FBS for 48 h before they were removed from the plates by trypsinization and counted manually.

Proliferation Studies and Antisense Adenoviral Infection

Cells were subcultured into six-well (proliferation studies) or 12-well (antisense adenovirus infection) culture plates at a density of 1 × 10⁴ cells/cm² and grown for 3 d in 10% FBS. Adenovirus with or without the antisense construct was then added to cultures at 10 or 100 pfu/cell for 24 h. This was followed by a 24-h period of quiescence in 1% FBS and then a 48-h exposure to either 10% FBS, 40 ng/mL PDGF, or 1% FBS. Cells were removed from the plates by trypsinization, and manual cell counts were performed. All treatments were done in triplicate, and means from individual experiments (patients) were pooled for statistical analysis.

Immunoblotting

For detection of PKC- by immunoblotting (8), control cells and cells infected with adenovirus (100 pfu/cell) were subcultured into 24-well culture plates. After infection, cells were stimulated with 40 ng/mL PDGF for 24 h and then extracted directly into SDS loading buffer (4% SDS; 15% glycerol; 62.5 mM Tris-HCl, pH 6.8; 0.005% bromophenol blue; and 200 mM dithiothreitol), then mixed 1:1 with water.

For electrophoresis, samples were resolved on SDS polyacrylamide (10%) gels using a Bio-Rad Mini Protean apparatus. Proteins were then electroblotted onto nitrocellulose membranes. Specific polyclonal antibodies directed against PKC-, PKC-, and PKC- were obtained from Santa Cruz Biotechnology.

Western blots were developed by the ECL system (Amersham Pharmacia Biotech, Little Chalfront, UK), using the isoform-specific primary antibody, and secondary antibody obtained from Sigma. Relative signal intensities were quantified by densitometry.

Analysis of Results

To obtain mean results from cells, which were derived from different patients and exhibited a wide range of growth rates, cell counts were expressed on a scale relative to that obtained in the presence of 1% FBS but in the absence of virus. Values were compared with the use of one-factor analysis of variance with repeated measures and Fisher's PLSD. Levels of significance were set at P < 0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

At a virus concentration of 100 pfu/cell, all cells were found to express high levels of GFP, although the levels of expression varied from cell to cell (23). Growth curves constructed for cells from the same patient stimulated with 10% FBS (19) in the presence of virus at 0 and 100 pfu/cell revealed that the increase in viable cell counts was not affected by 100 pfu/cell (Figure 1). Thus, concentrations of 10 and 100 pfu/cell were chosen for subsequent experiments.

View larger version (15K): [in this window] [in a new window]   Figure 1.   The effect of the presence of the adenoviral vector at a concentration of 100 pfu/ cell (squares) on cell growth compared with cells not exposed to virus (circles) in response to 48 h exposure to different concentrations of FBS. Values for manual cell counts are given for triplicate estimations. Vertical bars represent SEM.

In separate experiments, the presence of the antisense PKC- adenovirus at 10 and 100 pfu/cell significantly reduced proliferation produced by 48 h exposure to 40 ng/mL PDGF, compared with controls treated with no adenovirus or adenovirus expressing no transgene (Ad-MX17) (Figure 2). The amount of 10 pfu/cell appeared to produce a greater inhibition of growth, although the responses to 10 and 100 pfu/cell were not significantly different from each other.

View larger version (28K): [in this window] [in a new window]   Figure 2.   The effect of the presence of virus containing antisense PKC- at 10 (striped bars) and 100 (stippled bars) pfu/cell, together with control cells with no virus (open bars), cells incubated with virus containing wild-type PKC- (closed bars), and virus with no DNA insert (shaded bars), at 100 pfu/cell, on cell growth in response to 10% FBS or PDGF 40 ng/mL. Mean values for manual cell counts (from three experiments) are expressed relative to that for cells exposed to PDGF and no virus. Values for cells exposed to 1% FBS are also shown. *Significant difference from value for PGDF in the absence of virus, P < 0.05.

None of the concentrations of antisense virus used produced any effect on FBS-induced responses. In addition, infection of the cells with the adenovirus expressing wild-type PKC- (100 pfu/cell) produced no change in cell proliferation induced by 10% FBS or 40 ng/ml PDGF. Finally, expression of the antisense had no effect on cells made quiescent by incubation in 1% FBS, indicating that it does not have a nonspecific effect on cell viability. These results are shown in Figure 2. The relative mean cell counts after stimulation with PDGF in cells containing the antisense (100 pfu/cell), wild-type (100 pfu/cell), virus control with no insert (100 pfu/cell), and no virus were 37 ± 5, 67 ± 18, 102 ± 13 (mean values ± standard error of the mean [SEM]) and 100%, respectively. The corresponding values for cells stimulated with 10% FBS were 100 ± 22, 67 ± 24, 122 ± 18, and 85 ± 22%, respectively. The value for cells expressing the antisense and stimulated with PDGF was significantly different from each of the other treatment categories (n = 3, P < 0.05). Counts in cells containing the wild-type construct were not significantly different from control values.

Immunoblots revealed expression of PKC- protein in all cells, whether they were infected with viruses (at 100 pfu/cell) expressing wild-type PKC- or antisense PKC-, or virus with no construct. However, the density of the immunoblots was decreased by 47 ± 16% in cells that contained antisense PKC- viruses when compared with cells that contained no virus (Figure 3). Immunoblots for PKC- and PKC- , however, were not significantly decreased (88 ± 12 and 98 ± 22% of control, respectively; n = 3) in the presence of the PKC- antisense (Figure 3).

View larger version (26K): [in this window] [in a new window]   Figure 3.   Western blots for cells containing either no virus (control) or adenovirus expressing antisense PKC- (100 pfu/cell) exposed to PDGF, 40 ng/mL. Blots were developed for PKC-, PKC-, and PKC- from three separate experiments. (A) Western blots for PKC isoforms in control (C) and antisense-treated (AS) cells. (B) Mean densitometry results for the three PKC isoforms, with vertical bars representing SEM. *Significantly different from control value, P < 0.05.

	Discussion

Top Abstract Introduction Methods Results Discussion References

This study demonstrated that an antisense of PKC- can inhibit the proliferation of HASM cells stimulated with PDGF, but not proliferation induced by FBS. That the response is isoform-specific was borne out by the finding that the amount of protein for PKC- in cells expressing the antisense was reduced, whereas that for two other isoforms, one calcium-dependent and the other calcium-independentrespectively, PKC- and PKC- was not affected. In addition, our results demonstrate that adenoviruses can be successfully used to carry sense and antisense genes into HASM cells with high efficiency.

Our previous studies showed that, in HASM cells proliferating in response to PDGF or FBS, there was a significant increase in the amount of PKC- protein (9). Moreover, this did not occur for PKC-, -I, -II, -, - , - -, or - in response to the same stimuli. In addition, prostaglandin (PG) E₂, which inhibits proliferation (19), almost completely inhibited this increase in PKC- protein. The results of the present study increase the strength of the evidence for a causal relationship between PKC- and PDGF-induced mitogenesis in HASM cells and also show that the increase in PKC- is required for cell proliferation in response to PDGF rather than being a consequence of proliferation. The fact that FBS-induced proliferation was unaffected by the presence of the antisense indicates that PKC- is not a universal mediator of HASM proliferation and may be critical only in some tyrosine kinase receptor-mediated signal transduction pathways. In addition, in our previous study (9), although FBS and PDGF both produced increases in PKC- protein and both induced proliferation, the increase in PKC- in response to PDGF was demonstrable in both cytosolic and membrane fractions of the cells. This was not the case for FBS, which increased PKC- protein only in the cytosolic fraction. The absence of an increase in the active membrane fraction may explain the lack of effect of the PKC- antisense on FBS-induced proliferation in the current experiments.

In the present study, the antisense of PKC- reduced PDGF-induced mitogenesis by 63% compared with control. Thus, although there was significant inhibition of PDGF-mediated responses, mitogenesis was not completely abolished. It is likely that even though all cells may have been infected by the adenovirus, the levels of expression of the transgene varied from cell to cell (23). High levels of antisense expression may be necessary to achieve full inhibition. Lower levels of expression in some could lead to only partial inhibition, and this may be the most likely explanation for our findings.

The signal transduction events upstream and downstream of PKC- have been studied in a variety of cell types, but the placement of PKC- in these pathways remains unclear. In response to PDGF, PKC- has been reported to directly activate Raf-1 (25) or to be directly activated by Ras, which can also activate c-Raf. Activation of Ras is critical for PKC- activation (26), and phosphorylation by PIP3-dependent kinase (PDK) (27) has also been shown to activate PKC-. In pathways leading to gene expression, activated PKC- inactivates IB leading to regulation of nuclear factor-B (28). These pathways have not been defined in HASM cells and, as we (8) and others (29) have shown, distribution of PKC isoforms as well as their involvement in mitogenesis is markedly different in animal airways. Our antisense data in the present study would indicate that PKC- is not an essential component of FBS-induced proliferation and this may be because G-protein- coupled pathways are involved in this response. However, this would conflict with the findings of Ammit and colleagues (30), who reported that p21ras is a common step in mitogenic pathways arising from stimulation of both receptor tyrosine kinase and G-protein-coupled receptors.

It is difficult to speculate on the nature of pathways that could be independent of PKC- and thus account for the lack of effect of the antisense on FBS-induced proliferation. Whether FBS stimulates other kinases such as p70^S6K, which are independent of PKC- (31), is not known. Knowledge of the pathways leading to HASM proliferation is incomplete and marked specie differences abrogate extrapolation from animal models. We have shown in previous studies that PGE₂ and heparin both inhibit HASM mitogenesis (19), the former most likely via a cyclic adenosine monophosphate-dependent mechanism (24) and the latter by an as-yet-undelineated pathway. The events which occur distal to tyrosine kinase and proximal to cyclin D are likely to involve the MEK/ERK pathway. Recently, it has been reported that the GRB2/SOS/Ras/ Raf pathway and the PI3K/PDK-1/PKC- pathwayshitherto considered functionally separateare jointly required for ERK activation in rat adipocytes (32). Whether this holds true for proliferation in HASM cells requires investigation.

In the present study the PKC- antisense produced a marked decrease in the levels of PKC- protein as detected by immunoblotting, but did not decrease the protein level for two other isoforms, viz, PKC- and PKC- . This contrasts with the findings from some previous studies that used a dominant negative mutant of PKC- and found that it inhibited activated PKC- as well as PKC- (33). Use of the antisense approach in our study presumably avoids the potential for a dominant negative mutant to antagonize upstream substrates for other kinases.

The evidence from the current study for an association between PKC- and mitogenesis raises the possibility for selective inhibition of this isoform. Such an example already exists, in that inhibition of PKC- with a novel, orally active inhibitor ameliorates vascular dysfunction in a rat model of diabetes (34). Our study examined the relationship between PKC- and mitogenesis in HASM cells, but these were not obtained from patients with asthma. If it is possible in the future to demonstrate this relationship in asthmatic cells, then it would be conceivable that new pharmacologic or antisense approaches could be used to inhibit the ASM hyperplasia that is an unwanted feature of asthmatic airways.