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(Circulation Research. 1995;76:21-29.)
© 1995 American Heart Association, Inc.

Articles

Differentiation of Vascular Smooth Muscle Cells and the Regulation of Protein Kinase C-

Hermann Haller, Carsten Lindschau, Petra Quass, Armin Distler, Friedrich C. Luft

From the Department of Medicine and Nephrology, Steglitz University Hospital, and the Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, Rudolf Virchow University Hospitals, Free University of Berlin (Germany).

Correspondence to Hermann Haller, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany.

	Abstract

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Abstract Dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) are important features of atherosclerosis. The molecular mechanisms are largely unclear; however, protein kinase C (PKC) is a key enzyme in the intracellular signaling pathways that mediate this process. We studied the activity and immunoreactivity of PKC- in primary cultures of VSMCs from rat aortas under different conditions of growth and differentiation. PKC- was determined under the following conditions: (1) during the growth phase and after confluence of cultured (passages 1 through 3) VSMCs, (2) before and after induction of differentiation in VSMCs by retinoic acid, and (3) in primary cultures of VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats during early passages. PKC activity was measured by in vitro substrate phosphorylation. PKC- immunoreactivity was assessed by Western blot using specific polyclonal antibodies and by immunostaining with confocal microscopy. Cell proliferation was measured by direct count. The cell phenotype was characterized by immunostaining and Western blot for -actin and desmin. PKC- expression and PKC activity during VSMC growth showed a decrease during rapid growth and an increase in confluent cells. This pattern was associated with the respective changes in cell differentiation. Retinoic acid induced an increase in PKC- expression together with a more differentiated phenotype. Subcultured, rapidly growing VSMCs from SHR showed a decreased PKC- expression compared with cells from WKY rats. To establish cause and effect, we next microinjected either PKC- or inactivated material directly into dedifferentiated cells. We found that cells injected with active PKC- expressed increased amounts of actin compared with control cells. We identified a close correlation between PKC- and actin immunofluorescence. We conclude that PKC- is downregulated in rapidly growing VSMCs. Our findings demonstrate an inverse association between PKC- expression and VSMC differentiation. They suggest a role for downregulation of PKC- in the proliferative response of these cells.

Key Words: protein kinase C • protein kinase C isoforms • vascular smooth muscle cells • spontaneously hypertensive rats • cell proliferation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hypertrophy and proliferation of vascular smooth muscle cells (VSMCs) are both important in the development of chronic vascular disease.¹ In normal uninjured arteries, VSMCs exist in a nonproliferating differentiated state, the so-called "contractile" phenotype.² This phenotype is characterized by high levels of muscle-specific actins and intermediate filaments.² In particular, the expression of -actin has been used as a marker for the state of VSMC differentiation.^{3 4} The expression of these differentiated characteristics is altered in intimal thickening and in atherosclerotic lesions.⁵ This dedifferentiation, which occurs in response to growth factors and growth-promoting surfaces, also takes place when the isolated cells are subcultured in vitro.² The dedifferentiated state is also termed the "secretory" phenotype, because it is associated with loss of -actin and desmin and an increase in cell organelles involved with protein synthesis and secretion. Since the phenotypic changes appear to be a continuum, Chamley-Campbell et al² prefer the term "modulation" rather than dedifferentiation to describe this change in phenotype. The elucidation of phenotypic modulation is pivotal to the understanding of VSMC proliferation.

Isoforms of a phospholipid-dependent serine/threonine protein kinase system, also termed protein kinase C (PKC), are important in the modulation of cell phenotype.⁶ PKC isoforms are activated by a wide variety of growth factors and hormones^{7 8} and appear to be involved in mitogen signaling.⁹ Stimulation of PKC by phorbol ester induces VSMC proliferation,¹⁰ whereas inhibition of PKC decreases platelet-derived growth factor (PDGF)-induced proliferation.¹¹ Activity and expression of PKC isozymes during VSMC differentiation and dedifferentiation have not been delineated. One of the calcium- and phospholipid-dependent isoforms in VSMC is PKC-.^{12 13} We have recently observed that PKC- is translocated to the nucleus of VSMCs after stimulation of VSMC with mitogens.¹⁴ Thus, we tested the hypothesis that increased enzyme activity and expression of PKC- are associated with dedifferentiation and growth of VSMCs. We found that PKC- was downregulated when VSMCs assumed the secretory dedifferentiated state. When the cells reassumed their contractile state, PKC- expression reverted to normal. High intracellular protein levels of PKC-, generated by microinjection of the enzyme, induced actin expression and VSMC differentiation.

	Materials and Methods

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Phorbol ester DPB (12-deoxyphorbol 13-isobutyrate), histone type III-S, DEAE cellulose, retinoic acid (ß-all trans), and all other materials, if not stated otherwise, were purchased from Sigma Chemical Co. [³²P]ATP, ³²P, and the synthetic peptide were obtained from Amersham. 1,2-Diolein and phosphatidylserine were purchased from Avanti Polar Lipids.

Adult male spontaneously hypertensive rats (SHR), weighing 390±17 g with a mean tail-cuff systolic blood pressure of 175±15 mm Hg, and adult male Wistar-Kyoto (WKY) rats, weighing 378±16 g with a mean tail-cuff systolic blood pressure of 105±10 mm Hg, were obtained from Fisher Laboratories. Care of the animals was in accord with institutional and American Physiological Society guidelines.

VSMC Cultures
Rat aortic VSMCs were cultured by procedures modified from Chamley-Campbell et al.² Briefly, the rats (12 to 14 weeks) were killed instantly, and their thoracic aortas were excised. After adherent fat and connective tissue were removed, the aortas were cut longitudinally, and the endothelial cells were removed by gentle scraping with fine forceps. The aortas were then minced into small pieces and were incubated at 37°C for 2 hours in phosphate-buffered saline (PBS) without calcium but with 1 mg/mL collagenase (type I, 150 IU/mg, Worthington Biochemical Corp), 0.5 mg/mL elastase (type III, 40 IU/mg, Sigma), and 0.5 mg/mL trypsin inhibitor (Sigma). After 2 hours, Dulbecco's modified Eagle's medium (DME/F-12) containing 10% fetal calf serum (FBS, GIBCO) was added to the suspension to inactivate enzymes. The cells were then centrifuged at 120g for 10 minutes, and the pellet was resuspended in DME/F-12 with 10% FBS. The cells were then seeded at a density of 3 to 5x10⁵/cm² and were cultured at 37°C in 95% air/5% CO₂. Cells from passages 2 through 4 were used in all experiments. The phenotype of the cultured VSMCs was determined by staining the cells for -actin and desmin. Antibodies for muscle-specific -actin and desmin were obtained from Boehringer Mannheim.

Measurement of PKC Activity in VSMCs
PKC activity was measured in growing, nonconfluent (days 2 and 5), and confluent VSMCs (days 9 to 12) by using a modification of the procedures described by Heasley and Johnson.¹⁵ Growth medium was aspirated from the plates and was replaced with a solution containing (mmol/L) NaCl 137, KCl 5.4, Na₃PO₄ 0.3, K₃PO₄ 0.4, HEPES (pH 7.3) 20, MgCl₂ 10, ß-glycerophosphate 10, EGTA 7.5, CaCl₂ 2.5, KRTLRR peptide 600 (Bachem), and [-³²P]ATP 250 (20 mCi/mL), along with 50 mg/mL digitonin. The reaction was stopped after 15 minutes at 30°C with 10 µL of ice-cold 50% (wt/vol) trichloroacetic acid. With the peptide and ATP concentrations used, the rates of kinase activity were linear for 20 minutes. Aliquots (45 µL) of the reaction mixture were then spotted on phosphocellulose (Whatman P-81) and were washed six times for 10 minutes with 75 mmol/L phosphoric acid. The filters were then counted in a scintillation counter, and the results were expressed in counts per minute per microgram protein.

Immunoblotting
Cultured VSMCs were scraped off, resuspended in 1% sodium dodecyl sulfate (SDS), and immediately placed in boiling water as described previously.¹⁴ After estimating the protein content, 50 µg of protein was loaded onto each lane of 10% SDS–polyacrylamide gel. The samples were then electroblotted onto Immobilon-P membranes (Millipore). The membranes were successively incubated, first with blocking buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.5), 3% (wt/vol) bovine serum albumin (BSA), 0.2% (vol/vol) Tween 20, and 0.02% NaN₃ for 120 minutes at room temperature. The next incubation was conducted with isoenzyme-specific antibody diluted in incubation buffer containing 137 mmol/L NaCl and 20 mmol/L Tris-HCl (pH 7.5) at room temperature. We used highly specific monoclonal antibodies directed against rabbit brain PKC that reacted specifically with the subspecies of PKC (anti-PKC, type III, lot 12280, Upstate Biotechnology Inc [UBI]). For detection of immunoreactivity for the calcium-sensitive PKC isoforms , ß, and , a polyclonal antibody was raised in rabbits against a consensus sequence of the protein as previously described.¹⁶ A final incubation was carried out in TBS with biotinylated anti-rabbit or anti-mouse IgG (Amersham) and with streptavidin alkaline phosphatase peroxidase (Calbiochem) complex in incubation buffer. The membranes were thoroughly washed after each incubation with a buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.5), and 0.2% (vol/vol) Tween 20. Quantification of the protein bands was performed by densitometry on a video densitometer (model 620, Bio-Rad). The signals were then integrated, and the results were expressed in arbitrary units.

Immunocytochemistry and Confocal Microscopy
Immunocytochemistry and confocal microscopy were performed as described recently.¹⁴ VSMCs were fixed with 3% paraformaldehyde and were permeabilized with ice-cold 80% methanol. After incubation with 3% skimmed milk in PBS for 60 minutes, the preparation was incubated for 1 hour at room temperature with the PKC antibody (UBI) diluted in 0.1% BSA (1:80) or with antibodies against actin or desmin. The preparation was next washed three times with PBS and was then exposed to the secondary antibody (fluorescein isothiocyanate–conjugated anti-mouse IgG at 1:100 and 0.1% BSA/PBS, Pierce Chemicals) for 60 minutes. In the first experiments characterizing VSMC actin and desmin expression (Fig 1), rhodamin-conjugated antibodies were used, and immunofluorescence was measured with a Nikon epifluorescence microscope (Nikon Diaphot) with an excitation wavelength of 550 nm (Spex Industries Inc) and an emission wavelength of 580/590 nm. Exposure time was 30 seconds, and the different panels shown in Fig 1 were obtained under identical conditions.

View larger version (34K): [in this window] [in a new window]   Figure 1. Immunostaining (A and B) and Western blots (C and D) of subcultured vascular smooth muscle cells (VSMCs) during growth (days 2 and 5) and at confluence (days 9 to 12) for -actin (A) and desmin (B). For immunofluorescence, rhodamin-conjugated antibodies were used, and immunofluorescence was measured by use of a Nikon epifluorescence microscope (representative photomicrographs from five independent experiments). Growing cells (secretory phenotype) exhibited lower expression of these cytoskeletal proteins than did confluent cells (contractile phenotype).

For confocal microscopy, the preparation was mounted with 50% glycerol under a glass coverslip on a Nikon Diaphot microscope. An MRC 600 confocal imaging system (Bio-Rad Laboratories) with an argon laser was used. At least 10 to 18 cells from each of at least three independent experiments were examined under each experimental condition. Images were acquired in the normal scanning mode with a Kalman filter of 3 (indicates exposure time of 3 seconds). For each set of experiments, identical settings for power of the light source, confocal aperture, gain, and beam alignment were used. The results were reproduced by two separate investigators. The observers were unaware of the experimental design and antibodies used.

Quantification of the signal intensity of single cells was performed by using histogram/area functions (COMOS software). The cells were outlined manually, and the calculated mean fluorescence intensity was obtained for the delineated regions. The integrated fluorescence intensity was then divided by the delineated area. Data are presented as the mean fluorescence intensity per square micrometer.

Microinjection
Microinjection of VSMCs was carried out as described by Gressmann et al.¹⁷ PKC- (GIBCO) was dissolved in 20 mmol/L Tris buffer at a concentration of 1:10. For control experiments, PKC- was first heated (60°C for several minutes) and then dissolved in PBS (1:10 or 1:5). Microinjection was carried out with an Eppendorf microinjector. Active PKC- and denatured PKC- were injected at a concentration of either 20 or 10 µg/mL in 40 to 70 fL per cell. VSMCs were microinjected 2 days after plating. Before plating, areas on coverslips were marked by a diamond knife, and each cell within these areas was used for injection experiments and assessed later. To confirm the efficiency of the microinjection, VSMCs were stained 30 minutes after the procedure and stained for PKC- immunoreactivity. We observed a significantly increased immunoreactivity for both native and denatured PKC- after microinjection. The immunoreactivity was mostly located in the cytosolic region (data not shown).

Statistics
Statistical analysis was carried out on a Macintosh II computer (Apple Inc) by use of a commercially available statistical program (STATVIEW, Cricket Software Inc). The results (mean±SEM) represent duplicate measurements made on five to eight separate occasions. The n value given in the figures represents the number of separate experiments. Statistical analyses were performed with the nonparametric Wilcoxon test. Differences were considered to be significant at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PKC and Phenotypic Modulation of VSMCs
In the first set of experiments, subcultured VSMCs (passages 1 through 3) were seeded at a density of 3 to 5x10⁵/cm² and were grown in 10% FCS. Under these conditions, cells reached confluence within 9 to 12 days. Expression of -actin and desmin were used to characterize phenotypic changes of VSMCs. Fig 1 shows the immunostaining (panels A and B) and protein expression (panels C and D) for -actin (panel A) and desmin (panel B) at days 2 and 5 and in the confluent state. On days 2 and 5, the growing cells expressed low levels of -actin and desmin (Fig 1C and 1D). On day 2, the cells demonstrated a fibrillar pattern for both proteins (secretory phenotype) (Fig 1A and 1B). In contrast, confluent VSMCs (contractile phenotype) showed a significantly increased expression of both -actin and desmin. Actin was organized in straight, dense bundles of so-called "stress" fibers, which represent a more differentiated phenotype (n=5).

PKC activity and expression of PKC were measured after 2 and 5 days of growth and after the VSMCs reached confluence. In Fig 2, expression of all calcium-sensitive PKC isoforms (left panel) and PKC- (right panel) is shown. The differentiated contractile cells showed an increased expression of PKC and its isozyme compared with the dedifferentiated secretory cells. This increase in PKC- expression was also observed when immunostains of VSMCs for PKC- were examined with the confocal microscope (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 2. Expression of total protein kinase C (PKC) (left) and the isozyme PKC- (right) in proliferating and confluent vascular smooth muscle cells (VSMCs). The differentiated (contractile) cells show an increased expression of PKC and its isozyme compared with dedifferentiated (secretory) cells (n=5).

Fig 3 shows the PKC activity, expressed as protein phosphorylation, during days 2 and 5 of the growth phase and in differentiated confluent cells (days 9 to 12). Enzyme activity was low during the growth of VSMCs and was increased in differentiated (contractile phenotype) cells (n=6, P<.05). This increase in enzyme activity was accompanied by increased protein expression of both total PKC (Fig 2, left panel) and PKC- (Fig 2, right panel).

View larger version (23K): [in this window] [in a new window]   Figure 3. Bar graph showing protein kinase C (PKC) activity as measured by protein phosphorylation in growing (days 2 and 5) and confluent (days 9 to 12) vascular smooth muscle cells. PKC activity increased as the cells assumed the differentiated (contractile) phenotype (n=6).

Retinoic Acid
We then tested the effect of retinoic acid on VSMC differentiation and PKC expression. Subconfluent VSMCs (days 5 and 6) were incubated with retinoic acid (10^-9 mol/L) for 12 and 48 hours in 1% FCS. Retinoic acid caused a differentiation of the cells, which assumed a more contractile phenotype. We observed increased expression of -actin and PKC-, which was time dependent. Photomicrographs at the top of Fig 4 show the effect of retinoic acid on the immunostaining of -actin (upper) and PKC- (lower) by use of confocal microscopy after retinoic acid (10^-9 mol/L) exposure for 48 hours (right); control cells without exposure to retinoic acid are shown on the left. The increase in -actin stress fibers and the increase in PKC- expression, particularly in the perinuclear region, can be readily appreciated. In the graph shown at the bottom of Fig 4, the quantitative analysis of these experiments is shown. Retinoic acid induced a significant increase in the mean fluorescence of both -actin and PKC- expression (analysis of >60 cells in five separate experiments).

View larger version (21K): [in this window] [in a new window]   Figure 4. Top, Confocal photomicrographs show immunostaining for -actin (upper) and protein kinase C- (PKC-) (lower) after (right) retinoic acid (10-9 mmol/L) exposure for 48 hours; control cells without exposure to retinoic acid are shown on the left. Retinoic acid induced an increase in -actin stress fibers (upper) and an increase in PKC- expression, particularly in the perinuclear region. Bottom, Bar graph shows the quantitative analysis of these experiments. Retinoic acid induced a significant increase in the mean fluorescence of both -actin and PKC- expression (analysis of >60 cells in five separate experiments).

In Fig 5 is shown the PKC activity as measured by protein phosphorylation in VSMCs exposed to retinoic acid for 12 and 48 hours compared with control cells. Retinoic acid induced an increase in PKC activity over time (P<.05).

View larger version (28K): [in this window] [in a new window]   Figure 5. Bar graph showing protein kinase C (PKC) activity as measured by protein phosphorylation in vascular smooth muscle cells exposed to retinoic acid for 12 and 48 hours compared with control cells. Retinoic acid induced an increase in PKC activity (n=6).

VSMCs From Hypertensive Animals
In this set of experiments, expression of PKC- was compared in VSMCs from the aortas of SHR and WKY rats. VSMCs were isolated and subcultured. PKC- was measured in confluent cultures in different passages. Fig 6 shows the expression of PKC- in passages 1 through 4 of VSMCs from SHR and WKY rats. In all passages, PKC- expression was lower in VSMCs from SHR compared with VSMCs from WKY rats. Interestingly, PKC expression decreased continuously in subcultured VSMCs and almost disappeared by passage 4.

View larger version (22K): [in this window] [in a new window]   Figure 6. Protein kinase C- (PKC-) expression in subcultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (passages 1 through 4). Top, Results of Western blotting. Bottom, Graph showing relative absorption of the bands. PKC- expression of the cells was lower in SHR than in WKY rats. The expression decreased through time in both strains (n=4).

We next analyzed the growth response of VSMCs from both strains by exposing the cells to PDGF. As shown in Fig 7, the growth response of SHR VSMCs was significantly greater than the response from WKY VSMCs in all passages (P<.05). We also observed that the growth rate response to PDGF increased throughout the passages, indicating that increased growth rate response and PKC- expression (Fig 6) were associated inversely.

View larger version (49K): [in this window] [in a new window]   Figure 7. Bar graph showing platelet-derived growth factor–induced proliferation of vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at passages 2 through 4. The growth response rate was greater in SHR than in WKY rats and also showed an increase between passages 3 and 4 (n=8).

Microinjection of PKC- Into VSMCs
In the last set of experiments, we investigated whether or not an increase in intracellular PKC- concentration would induce actin expression and differentiation in cultured VSMCs. We microinjected 40 to 70 fL of recombinant PKC- at a concentration of 20 or 10 µg/mL into subconfluent VSMCs 2 days after plating. Microinjection of denatured PKC- was used as a control. VSMCs were then stained for actin and PKC- immunoreactivity after 12 and 24 hours. Fig 8 shows the results of the microinjection experiments with inactivated PKC- (10 µg/mL) (top panels, control) and active PKC- (10 µg/mL) (bottom panels) after 24 hours. Confocal photomicrographs with a stain for actin (green) and PKC- (red) and simultaneous staining are shown (representative of three independent experiments). The injection of VSMCs with active PKC- resulted in the appearance of abundant actin fibers compared with control VSMCs. PKC-–injected VSMCs also showed a higher expression of PKC- after 24 hours compared with the control cells.

View larger version (0K): [in this window] [in a new window]   Figure 8. Confocal photomicrographs showing the results of microinjection experiments with inactivated protein kinase C- (PKC-) (upper) and active PKC- (lower) with a stain for actin (green) and PKC- (red) and simultaneous staining. The injection of active PKC- resulted in the appearance of abundant actin fibers compared with control (inactivated PKC-) (representative of three independent experiments).

We then analyzed the relation between PKC- immunofluorescence and actin expression in the microinjected cells. In Fig 9, the correlation between PKC- fluorescence intensity (abscissa) and actin fluorescence intensity (ordinate) is shown (showing 52 cells from three independent experiments). Open circles indicate control cells microinjected with denatured PKC-; closed circles represent cells microinjected with active PKC-. A highly significant correlation between the two variables was identified.

View larger version (10K): [in this window] [in a new window]   Figure 9. Scatterplot showing the correlation between protein kinase C- (PKC-) fluorescence intensity (abscissa) and actin fluorescence intensity (ordinate) (52 cells from three independent experiments). Open circles indicate control cells microinjected with denatured PKC-; closed circles, cells microinjected with active PKC-. A highly significant correlation between these two variables was identified.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results showed that the activity and expression of PKC- VSMCs were significantly lower in dedifferentiated VSMCs compared with quiescent confluent VSMCs. Furthermore, differentiation of VSMCs induced by retinoic acid was accompanied by an increase in both PKC- expression and PKC activity compared with control VSMCs. Faster growing VSMCs from SHR showed a decreased expression of PKC- compared with VSMCs from WKY rats. Finally, microinjection of PKC- into VSMCs induced an increase in actin expression and led to a more differentiated phenotype of the VSMCs. These findings argue that levels of PKC- expression play a role in the differentiation-dedifferentiation process of VSMCs.

Although the relation between PKC expression and cell differentiation has not previously been studied in VSMCs, PKC was found to play a role in several other cell types.¹⁸ For instance, in the HL-60 cell line, a frequently used model for the investigation of cell differentiation, differentiation could be induced by phorbol ester, which activates PKC.¹⁹ Moreover, HL-60 cells with a defective PKC pathway did not differentiate in response to phorbol ester compared with cells with no such defect.²⁰ Furthermore, in an earlier study Makowske et al²¹ showed that in HL-60 cells, expression of the PKC isoforms , ß, and increased during differentiation. In addition, an increase in PKC- during differentiation was also observed in hemopoietic cells.^{22 23} The increase in PKC expression in that particular study was regulated at the level of mRNA. Finally, an increase in PKC expression during the process of differentiation was also demonstrated in PC12 cells,²⁴ keratinocytes,²⁵ and epidermal tissue.²⁶

We also found that differentiated VSMCs had higher levels of PKC activity compared with dedifferentiated VSMCs. A similar increase in PKC activity during differentiation has been observed in keratinocytes.²⁵ An increased PKC activity was also observed in differentiated T lymphocytes by us (unpublished observations) and others.²⁷ Taken together, the evidence suggests that the process that moves cells toward differentiation (the contractile phenotype) is accompanied by an increase in PKC expression and PKC enzyme activity. On the other hand, moving to the dedifferentiated (secretory) phenotype appears to be associated with a decrease in PKC- expression and enzyme activity. However, the role of PKC in cell differentiation may nonetheless depend on the cell type. For instance, neuroblastoma cells exhibited a decrease in PKC- during differentiation.²⁸

Our results show that expression of PKC- increases in confluent cells. This finding is compatible with an induction of PKC- expression by an increase in cell density or by cell-cell contact. Blank et al³ have shown that induction of differentiation markers in VSMCs is induced by cell-cell contact and can be dissociated from cell cycle withdrawal. This finding is important, since most studies of PKC regulation in other cell types have been carried out in confluent cell cultures.^{7 9 24 25 29 30 31} It is possible that some of the divergent findings involving PKC isozyme expression in the literature are due to this phenomenon.

We observed an increase in PKC- when VSMCs were exposed to retinoic acid for 48 hours compared with control cells not exposed to retinoic acid. Retinoic acid causes various cell types to differentiate.³² Our observations indicate that this effect of retinoic acid also applies to VSMCs and facilitates their assumption of the contractile phenotype. This conclusion is supported by the increased expression of -actin as well as the increase in "stress fibers" produced by the cells exposed to retinoic acid. Our experiments support the finding that PKC- expression is increased in differentiated compared with dedifferentiated VSMCs. As mentioned above, induction of differentiation in HL-60 cells by retinoic acid was accompanied by an increase in PKC- expression.³³ These cellular effects of retinoic acid are in part directly mediated by PKC.^{34 35 36} Oka et al²⁹ have observed that melanocytes treated with retinoic acid promptly differentiate and increase their PKC- expression, both at the protein and mRNA level. It is also possible that the increase in PKC activity induced by retinoic acid is mediated by other effects. For instance, retinoic acid prevents oxidative modifications in PKC.³⁷

In our next set of experiments, we observed that in the more rapidly growing SHR VSMCs, PKC- expression was lower than in the slower growing WKY cells. Interestingly, we also found that PKC- expression decreased with increasing passages of our VSMC cultures. This observation indirectly supports our hypothesis that PKC expression is decreased in dedifferentiated compared with differentiated VSMCs. The fact that VSMCs gradually lose their phenotypic properties during the first few passages is well recognized.^{2 4} Moreover, the observation that SHR VSMCs grow more rapidly in response to growth factors than do WKY VSMCs is also well established.^{38 39 40} However, the molecular mechanisms that are responsible for this enhanced growth response in SHR are still not clearly elucidated.⁴¹

To establish cause and effect, we next microinjected either PKC- or inactivated material directly into growing VSMCs with very low levels of PKC- expression. The microinjection led to a significant increase of PKC- protein levels in these cells. We found that cells injected with active PKC- expressed increased amounts of actin compared with control cells. This finding suggests that increased expression of PKC- is not only associated with differentiation of VSMC but also plays a distinct role in the differentiation process. It is presently unclear how the increase in PKC- induces actin expression. We cannot rule out the possibility that the effect of PKC- on actin expression is not direct but is mediated by secondary mechanisms, such as enhancement of transforming growth factor-ß. Interestingly, in the microinjection experiments, PKC- immunoreactivity was still increased after 24 hours in the VSMCs. Since the PKC- immunoreactivity was higher at 24 compared with 12 hours, it is possible that microinjection of PKC- enhances the expression not only of actin but also of PKC- itself.

Our observations demonstrate an association between the lower expression of PKC- and increased growth response. We cannot necessarily conclude from our data that a cause and effect relation between growth and PKC expression exists. However, the association was present when SHR and WKY rats were compared, as well as when early and late passages of VSMCs were compared. We believe that such a link is most intriguing. Several other groups of investigators have found that enhanced growth is associated with decreased expression of PKC.^{42 43} Furthermore, it has also been shown that overexpression of PKC in rapidly growing epithelial cells decreases the growth of the cells.⁴⁴ In NIH 3T3 cells, Mischak et al³⁰ described overexpression of PKC isoforms that leads to cell differentiation and a decrease in growth rate. However, they observed that different PKC isoforms could exert both negative and positive effects on growth in these cells. Thus, on the one hand, activation of PKC is an important signal in the messenger system leading from receptor activation to cell growth; on the other hand, growth itself appears to be associated with decreased protein levels of the enzyme as well as decreased PKC activity.

One possible explanation for these divergent findings may be that PKC is transiently activated by growth factors and subsequently is downregulated by a negative-feedback mechanism. In support of this notion is the fact that activation and translocation of PKC by phorbol ester is rapidly followed by protein breakdown of the enzyme. Therefore, not only initial activation but also subsequent downregulation may be important signals in cellular growth. In accord with this hypothesis, Berry and colleagues^{45 46} have demonstrated that a transient activation of PKC is not sufficient to elicit a growth response in T lymphocytes⁴⁵ and have argued that downregulation of PKC may be required for a growth response to occur.⁴⁶ Recently, Brooks et al³¹ showed that the downregulation of PKC and not its transient activation correlates with cell growth in melanocytes. Thus, our finding of decreased PKC- expression in VSMCs from SHR suggests that this decreased activation of PKC may play a role in the enhanced growth response of these cells.

In summary, we observed that PKC activity and the expression of the main calcium- and phospholipid-dependent PKC isoform in VSMCs, PKC-, are downregulated in dedifferentiated (secretory) VSMCs. Furthermore, PKC expression increased during the differentiation process to the contractile phenotype. We then demonstrated that in rapidly growing VSMCs, PKC- expression is less abundant than in more slowly growing VSMCs. Finally, we demonstrated that an increase in PKC- by microinjection induced the expression of actin and led to a more differentiated VSMC phenotype. Given the intricate balance between differentiation and growth in VSMCs, our results indicate that an increase in PKC- expression induces VSMC differentiation and that the downregulation of PKC- during dedifferentiation is one of the intracellular mechanisms that is important for further progression through the VSMC cycle. According to this notion, PKC would be initially activated by growth factors and subsequently downregulated by negative feedback. Indirect support for a dual role of PKC in cell proliferation has been reported by others.^{36 47} Further experiments are required to define the specific role of PKC- during the cell cycle of VSMCs and the function of other PKC isozymes in this process.

	Acknowledgments

 
This study was presented in part at the Annual Meetings of the American Society of Nephrology, 1992. The project was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Ha-1388/2-3).

Received January 21, 1994; accepted September 16, 1994.

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