Differential expression of protein kinase C isoforms in the human placenta

Placenta

(1996),

17, 461-469

Differential

Expression

of Protein

Kinase

C lsoforms

in the Human

Placenta

A. L. Ruzyckya, Department

of Obstetrics,

a Department Paper

T. Janssonb

of Obstetrics,

accepted

19

March

Gynecology Gynecology

and N. P. lllsleyC and and

Reproductive Reproductive

Sciences, Sciences,

University University

of California, of

Pittsburgh,

San

Francisco,

Pittsburgh,

CA, PA,

USA USA

1996

The extensive role played by protein kinase C (PKC) in signal transduction prompted this study of the expression and localization of PKC isoforms in human placental syncytiotrophoblast. -Membranes prepared from these cells and samples of villous tissue were analysed by immunoblotting and immunocytochemistry using isoform-specific antibodies. PKC /32, y, E and c were found to be present in both microvillous and basal membranes from term placenta. The a isoform was observed only on the basal membrane while the pl isoform was confined to the microvillous membrane. The basal : microvillous ratios for p2, y, E and [ ranged between 0.3 and 0.5, demonstrating a substantial asymmetry in plasma membrane localization. Immunocytochemistry supported the isoform identification and localization observed in the immunoblotting experiments. Moreover the cellular distribution showed that the majority of syncytical PKC was bound to the plasma membranes, in contrast to the other villous cell types. Immunoblotting experiments demonstrated significant increases in PKC p2 and E on the microvillous membrane and PKC y and E on the basal membrane between 16 and 40 of weeks gestation. This is the first detailed mapping of PKC isoform distribution in an epithelial cell type and demonstrates the potential for selectivity in signal transduction through phosphorylation of isoform-specific and spatially-separated substrates. 0 1996 W. B. Saunders Company Ltd Placenta (1996), 17, 461-469

INTRODUCTION Protein kinase C (PKC) forms a pivotal point in a transmembrane signal transduction chain found ubiquitously in mammalian cells. It is commonly associated with a receptor/G protein-mediated pathway, and is activated by one or more of the second messengers produced by this pathway, which include phosphatidylserine (PS), diacylglycerol (DAG) and calcium (for reviews, see Azzi, Boscoboinik and Hensey, 1992; Nishizuka, 1992; Stabel and Parker, 1991). The activation by different combinations of these agents forms the basis of PKC isoform classification. At least 11 isoforms of PKC have been identified to date, categorized as the classical group (cPKC; a, PI, J32,y) requiring PS, DAG and calcium for activation, the novel group, nPKC; 6, E, q, 0) requiring PS and DAG, and the atypical group (aPKC; 5, h, l.t). Activation factors for the latter group are not yet clear, but they require neither diacylglycerol nor calcium (Johannes et al., 1994; Kochs et al., 1993; Nakanishi, Brewer and Exton, 1993). The distribution of Presented in part at the 41st Investigation, Chicago 1994 b Current address: Thomas natal Physiology, Department Medicinargatan 11, ~413 90 c To whom correspondence Obstetrics and Gynecology, 185 South Orange Avenue, 0143S4004/96/070461

Meeting of the Society for Gynecologic (Abstract No. P193). Jansson, M.D., Ph.D., Division of Periof Physiology, University of Goteborg, Goteborg, Sweden. should be addressed at: Department of UMDNJ-New Jersey Medical School, Newark, NJ 07103-2714, USA.

+ 09 $12.00/O

PKC isoforms, both in type and quantity, has been shown to differ between tissues; PKC isoform expression has been described in epithelial tissues (Knox, Johnson and Gordon, 1993; Leibersperger et al., 1991; Lin and Bazan, 1992), but the intracellular distribution has not been determined. In addition to the differences in tissue and cellular distribution, the various isoforms differ in their substrate specificities, either phosphorylating alternative substrates or displaying a differential activity towards the same substrate (Azzi, Boscoboinik and Hensey, 1992; Ido et al., 1987; Marais et al., 1990). There is abundant evidence of the involvement of PKC in syncytiotrophoblast function. PKC has been implicated in hormonal synthesis and release, including the production of progesterone (Kato et al., 1989), the secretion of hCG (Iwashita et al., 1992), the release of hPL (Wu and Handwerger, 1992), TGF-P (Ritvos and Eramaa, 1991) and IL-6 (Neki et al., 1993). PKC-mediated phosphorylation has also been observed in the regulation of cytoskeletal elements (Kenton, Johnson and Webb, 1989) and amino acid transporters (Kulanthaivel et al., 1991; Ramamoorthy et al., 1992). It is clear that the cellular content, location and isoform distribution of PKC is important information for the understanding of cellular regulatory functions. Two groups have assayed supernatants from human placental homogenates for the presence of the cPKC isoforms. Tertrin-Clary et al. observed calcium-stimulated activity in their samples, identifying the IX 0

1996 W. B. Saunders

Company

Ltd

462

Placenta

and p but not the y isoform (Tertrin-Clary et al., 1990; Tertrin-Clary, Chenut, and de la Llosa, 1991). Similarly, Nomura et al. identified the c1and p isoforms and showed a difference in the expression of a and p between the first and second trimester (Nomura et al., 1991); they also were unable to detect the y isoform. Although the syncytiotrophoblast is the dominant cell type in the placenta at term, the presence of other cell types such as fibroblasts, cytotrophoblast and endothelial cells precludes definitive assignment of the isoform types, quantitation and gestational development to a specific cell type. Moreover the use of supernatants from placental homogenates does not take account of the well-characterized partitioning of PKC between the cytoplasm and plasma membranes, the latter fraction containing the active form of the enzyme. In this report we have for the first time identified and quantified the PKC isoforms in an epithelial cell system, the human placental syncytiotrophoblast. The large, multinucleate nature of syncytiotrophoblast cells precludes isolation of significant numbers of these cells. We, therefore, chose to determine the distribution of PKC isoforms between the opposing faces of this epithelium using a membrane isolation procedure which generates matched and purified microvillous and basal membranes. Isoform expression and cellular distribution were also studied by immunocytochemistry in chorionic villous tissue. Finally, we examined the gestational development of PKC isoform expression and distribution over the second and third trimesters of pregnancy. MATERIALS Tissue

AND

acquisition

METHODS and preparation

Human placental tissue was obtained in accordance with guidelines approved by the Human Research and Biosafety Committees at U.C.S.F. Tissue was obtained from normal term deliveries (3841 weeks gestation), preterm deliveries (27-37 weeks gestation) and pregnancy terminations (16-22 weeks gestation). Preterm tissue was obtained from premature deliveries which were notable only for prematurity. The termination tissue was obtained from pregnancies terminated for social reasons, which showed no complications at the time of the procedure. The tissue was obtained within 30 min of delivery and cooled to 4°C before further processing. Placental tissue was used either for the preparation of microvillous and basal plasma membrane vesicles or for immunocytochemistry. Microvillous (MVM) and basal (BM) membrane vesicles were prepared by a modification (Jansson, Wennergren and Illsley, 1993) of a method described previously by Illsley et al. (Illsley et al., 1990). In brief, placental tissue was homogenized in 250 mM sucrose, 10 mM Hepes/Tris pH 7.4 at 4°C and subjected to differential centrifugation to isolate a high-speed membrane fraction. This fraction was treated with MgCl, to precipitate non-MVM. The supernatant fraction was used to prepare microvillous vesicles while the precipitate was resuspended and centrifuged through a sucrose density step

(1996),

Vol. 17

gradient to produce the BM fraction. These membrane fractions were highly enriched and showed minimal contamination with intracellular membranes or plasma membranes from non-syncytial cells (Illsley et al., 1990). -MVM and BM were stored at - 80°C. Blocks of tissue for immunocytochemistry (- 1 cm2) were cut from the placenta, close to the chorionic plate, avoiding connective tissue and vessels where possible. The tissue blocks were placed in 4 per cent paraformaldehyde, 100 mM phosphate, pH 7.4 at 4°C for a minimum of 24 h.

lmmunoblotting Placental MVM and BM obtained from the same tissue were solubilized in sodium dodecyl sulphate (SDS) and subjected to electrophoresis on 7.5 per cent polyacrylamide gels (PAGE) (30 pg of membrane protein/lane) by the method of Laemmli (Laemmli, 1970). After electrophoresis, proteins were transferred electrophoretically on to nitrocellulose membranes at 85 V for 24 min. Nitrocellulose membranes were incubated with 10 per cent normal goat serum (NGS)/O. 1 per cent Tween in Tris-buffered saline (TBS) for 1 h at room temperature to saturate nonspecific binding sites. The membranes were then incubated with peptide-derived, isozyme-specific antisera for 3 h at room temperature. After three washes with 0.1 per cent Tween in TBS, blots were incubated for 1 h with horseradish peroxides (HRP)-conjugated goat anti-mouse or anti-rabbit IgG in TBS. Protein bands of interest were detected calorimetrically using a high sensitivity Enzygraphic Web (IBI, New Haven, CT, USA) according to the manufacturer’s instructions. Detection of the specific primary antibody employed an amplified HRI-conjugate detection system. Development of the bands of interest occurred within 1 min using the enhanced HRP detection protocol. After optimal reaction time (development times were empirically optimized for the bands of interest), the blots were photographed using Polaroid type 55 positive/negative film. The position of the bands in the MV-M and BM samples were compared with control samples obtained from rat brain. Immunoreactive bands were quantitated from the photographic negatives using a GS300 scanning densitometer (Hoefer, San Francisco, CA, USA) interfaced to a computer, Isoform ratios at term (Table 2) and isoform changes over gestation (Tables 3 and 4) were analysed by parametric hypothesis testing using the Student’s t distribution (Lentner, 1972), while isoform ratio changes over gestation (Table 5) were analysed by StudentNewman-Keuls test. In experiments where the PKC isoform types were determined, equal amounts of protein (from the same sample) were loaded on to adjacent lanes of a gel, separated by SDS-PAGE and transferred to nitrocellulose. After blocking, individuals lanes were cut out and incubated with different PKC isoform antibodies as per the protocol above. Individual antibodies were used at dilutions found to combine linearity with maximum signal specificity and approximately equivalent staining intensity across the range of isoforms. Individual nitrocellulose

Ruzycky,

Jansson

and Illsley:

Expression

of Protein

Kinase

C in Human

Placenta

lanes from the same gel were recombined before washing, exposure to secondary antibody and development, enabling comparison after staining and densitometry. For experiments in which BM : MVR/I ratios for specific isoforms were obtained, equal amounts of protein from BM and MVM from an individual tissue sample were loaded side-by-side and treated identically throughout the analysis procedure. Gestational age comparisons were made by measuring isoform staining patterns for preterm samples relative to a standard set of term samples present on each gel.

463

Gibco-RBL (Gaithersburg, MD, USA). Electrophoresis equipment and reagents were obtained from Bio-Rad (Richmond, CA, USA), and the Enzygraphic Web Western detection system from IBI (New Haven, CT, USA), Araldite and paraformaldehyde were obtained from SPI (West Chester, PA, USA) and Ted Pella Inc (Redding, CA, USA), slides and propylene oxide from Fisher Scientific (Pittsburgh, PA, USA), diaminobenzidene and all other chemicals from Sigma Chemical Company (St Louis, MO, USA).

RESULTS lmmunocytochemistry

The loose, flexible nature of chorionic villous tissue makes cryosectioning inappropriate, while paraffin sections tend to show sub-optimal labelling characteristics. We elected, therefore, to use a plastic embedding technique which allows a high degree of labelling whilst maintaining tissue structure as described previously (Jansson, Wennergren and Illsley, 1993). Tissue was washed in 150 m-M sodium cacodylate buffer, dehydrated in graded ethanol, then the tissue was incubated in three changes of propylene oxide and transferred to a 1 : 1 mixture of propylene oxide : Araldite for a 2 h incubation followed by overnight infiltration with Araldite. Using polyethylene capsules, tissues were embedded in Araldite and cured at 60°C for 48 h. Semi-thin sections (1 pm) were cut and mounted on gelatin-coated slides. The plastic was etched by treatment with saturated potassium hydroxide in ethanol for 6 min. Slides were rinsed with ethanol, postfixed with 4 per cent paraformaldehyde, 100 mM phosphate buffer (10 min), incubated in 50 mM glycine and subsequently treated with 2.5 per cent H,Oz (5 min). Each of these steps was followed by washing (4 X 5 min) in TBS. The slides were blocked in 3 per cent NGS/5 per cent non-fat milk in TBS for 30 min at room temperature then incubated overnight at 4°C in a humidified chamber with primary antibody, diluted in 3 per cent NGS/ 5 per cent non-fat milk in TBS. Appropriate controls with normal rabbit serum or rabbit IgG were included to determine nonspecific staining. Sections were incubated with secondary (goat anti-rabbit IgG) and tertiary (rabbit peroxidase antiperoxidase IgG) antibodies (1 : 25-l : 100) for 30 min at room temperature. These incubations were preceded by blocking in 3 per cent NGS/5 per cent non-fat milk in TBS and followed by washing in TBS. Slides were treated with 0.05 per cent (w/v) 3,5-diaminobenzidine, 0.01 per cent (v/v) H,O, in TBS to visualize the bound primary antibodies. After the appearance of brown reaction product, slides were washed, dehydrated in graded alcohols, cleared in xylene and mounted. Slides were visualized by light microscopy. Materials

Anti-PKC antibodies were obtained from R & D Antibodies (Richmond, CA, USA), rabbit peroxidase anti-peroxidase from Organon Technica (Durham, NC, USA) and goat serum from

PKC

blotting

specificity

MVM and BM from normal term placental tissue were blotted and probed for the presence of the PKC ~1,fll, p2, y, 6, F and [ isoforms by immunoblotting. Polyclonal IgG antibodies generated against synthetic peptides corresponding to unique sequences of individual PKC isoforms were used to identify isoform expression (Table 1). According to the commercial source of the anti-PKC antibodies employed in this study, cross-reactivity of antibodies between isoforms is ~0.1 per cent (R & D Antibodies, Richmond, CA, USA). Specific isoform expression was confirmed on Western blots by determination of the appropriate, apparent molecular weight by comparison with molecular weight markers and by comparison with the immunoreactive bands observed in samples of rat brain cytoplasmic extracts containing the PKC isoforms (AcevedoDuncan et al., 1989). Figure 1 shows a typical blot of matched MVM and BM samples from three gestational ages, probed with antibody specific to PKC E. The apparent molecular weights of the immunoreactive bands in the placental samples are similar to, although slightly higher than, that of the rat brain control. In this example, both the microvillous and basal samples blotted for PKC E showed evidence of more than one band. In other samples blotted for PKC E or the other isoforms, the presence of multiple bands similar to those in Figure 1 was variable and generally less marked.

MVM

and

BM

isoform

profiles

The results obtained using paired MVM and BM from a minimum of three separate placental preparations, are shown in Table 2. They show the presence in both membranes of the p2, y, E and c isoforms. By contrast, only MVM samples contained the pl isozyme while the a isoform was confined to BM. The apparent molecular weights of the placental isoforms are shown in the second column of Table 2 compared with literature values. A close correspondence was observed between the apparent molecular weights obtained in these experiments and literature values. After quantitation by scanning densitometry, it was possible to compare the quantities of the isoforms common to both membranes. The last column in Table 2 shows the BM/MVM ratio for PKC p2, y, E and [. It is apparent that, when measured on a per microgram of

Placenta (1996), Vol. 17

464 Table

1.

Protein kinase C (PI(C) antigen sources

PKC Isoform

Source

Sequence location

Amino acids

0.

Human, rat, bovine Rat Rat Human, rat, bovine Rat Rat Rat

3133326 661-671 660-673 306-3 1%Cys 662-673 728-737 480492

WAGNZKVISPSEDRRQC SYTNPEFVINV SFVNSEFLKPEVFS NYPLELYERVRTGC SFVNPKYEQFLE FSYFGEDLMP YINPLLSAEESV

s2 Y 6

PKC

MVM

BM

MVM

BM

MVM

BM

MVM

BM

1 . Immunoblotting of protein kinase C (PKC) in placenta: expression of PKC E in microvillous (MVM) and basal (B-M) membrane fractions of human placenta. Equivalent amounts of paired placental sample protein (35 pg) were loaded in consecutive lanes on SDS-PAGE gels. Immunoreactive bands were Figure

observed at an apparent molecular weight of 77 kDa. Left to right: 19 week MVM; 19 week BM; 21 week MVM; 21 week BM.

Table

2.

PKC isoform profiles for microvillous membranes (B-M)

PKC rat brain

(MV.M) and basal

PKC isoform

Apparent molecular weight, kDa (lit. valueTef)

MVM

BM

BMMV-M ratioa

0.

76 (7Sb)

-

+

-

/ii1 ii

77 78 (77’) (77”) 76 83 (78’) (78’) 77 (84’) 74 (76b)

+ +-

++

0.41 - zk 0.09 (5) 0.30 5 0.07 (4)

+ +

+ +

0.35&0.11 (6) 0.47 f 0.14 (6)

a Ratios are given as mean & SEM; number of BM:MV-M pairs from separate placental preparations is given in parentheses. All ratios significantly less than 1.0 (P
membrane protein basis, the MVM contained substantially more of all the common isoforms. The values of the mean BM : MVM ratios for the common isoforms are all significantly less than unity (WO.05) but not significantly different from each other.

lmmunocytochemistry

The semi-thin plastic sections of chorionic villous tissue were stained for the PKC isozymes as described in the Methods section. The strongest staining was observed with the p2 and c antibodies. Figures 2(A) and (B) show typical sections stained for the PKC p2 isoform; for comparison, Figure 2(D) shows a villous tissue section stained using toluidine blue to illustrate

extract;

term MVM;

term BM;

16 week MVM;

16 Week

BM;

19 week MVM;

villous morphology. In Figures 2(A) and (B) strong microvillous staining for PKC p2 is apparent but there is little or no staining observable on the BM. In addition to the signal on the MVM, the capillary endothelium and other non-syncytial areas were also positive for PKC 82. Staining in the cells of the villous core was much more diffuse, suggestive of a substantial degree of cytoplasmic localization. The section in Figure 2(C) was stained for PKC 6; the distribution was similar in many respects to that in Figures 2(A) and (B) although the villous core staining was somewhat lessintense. Staining for the u and E isoforms was essentially negative while the pl and y isoforms were positive at an intermediate level (not shown). In terms of distribution, all those isoforms that showed positive staining gave similar results; there was moderate to intense staining on the MVM and minimal staining along the B-M. Of note was the lack of staining for any of the PKC isoforms in the syncytiotrophoblast cytoplasmic space. There was however clear cytoplasmic staining in non-syncytial cells. On a few of the sections it was possible to see some weak staining with the PKC CI isoform.

Gestational

development

In the investigation of gestational development, tissue was classified into one of three gestational age groups, termination (range 16-22 weeks gestation), preterm (27-32 weeks) and term (3841 weeks). For each isoform, samples from termination and preterm groups were compared on the same blots with a standard set of term samples. After densitometry, the term group was assigned the value of 100 per cent and the termination and preterm samples were quantitated as a percentage of the term value. The results of the measurements for MVM are shown in Table 3. It is apparent that there were

Ruzycky,

Jansson

and Ill&y:

Expression

of Protein

Kinase

C in Human

Placenta

465

Figure 2. Immunocytochemistry of protein kinase C (PKC) in placenta. Immunocytochemical detection of PKC isoforms in semi-thin sections (1 pm) of normal term human placental villous tissue. (A)-(C): Sections incubated with anti-PKC p2 [(A) and (B)] and anti-PKC < (C) antibody (1 : 50 dilution) followed by incubation with goat anti-rabbit IgG (1 : 25) and finally with rabbit peroxidase anti-peroxidase (1 : 25). Binding was visualized by incubation with 3,5-diaminobenzidine. Control sections incubated with normal rabbit serum instead of the specific anti-PKC antibodies were negative. Final magnification x 540; bar 25 pm. Arrow, fetal capillary; arrowhead, microvillous membrane. (D) Section stained with 1 per cent toluidine blue. Arrow, fetal capillary; arrowhead, syncytiotrophoblast cell layer.

significant increases in the microvillous content of the /32 and E isoforms towards term, although the termination and preterm levels were at the most, only 25 per cent below those at term. The results for measurements made using the BM are shown in Table 4. The a was not observed consistently in the termination or preterm BM samples, while the y isoform was not apparent in BM samples from either termination or preterm groups. The levels of E and < in the preterm period were significantly lower than term, while the 82 isoform did not show significant changes. As a result of the isoform changes described in the separate membranes, the BM : MVM

ratios for the common isoforms also showed changes over gestation (Table 5), with the p2 ratio rising from 0.35 in the preterm period to 0.89 in mid-gestation and falling to 0.41 at term. The ratio for the t: and 5 did not change significantly from mid-gestation to term.

DISCUSSION The goal of this research was to identify the isoforms of PKC in the human placental syncytiotrophoblast, to determine their

466 Table

Placenta

3. Protein kinase C (PKC) isoform profiles for microvillous membranes during development

PKC isozyme

Per cent of term value Termination Preterm (16-22 weeks) (27-32 weeks)

0.

-

;:

108 83 Zt It 20 10 (3) (lO)b

110 79 f 912(4)(3)

Y 6

112+23

110 + 29 (7)

(8)

80 & 8” (5) 1351.21 (7)

75 i 11” (5) 119 It 12 (11)

Values are expressed as a percentage of the value for microvillous membranes at term (mean Z!YSEM). Number of measurements are shown in parentheses. “Value less than term, WO.05; bvalue less than term, P
4. Protein kinase C (PKC) isoform profiles for basal membranes during development

PKC isozyme

Percent of term value Termination Preterm ( 16-22 weeks) (27-32 weeks)

0.

-

;: Y 6

-

i”

87 + 21 (5) 106 + 22 (11)

77 * 14 (4)

136 =t 17 (3) 72 5 9 (3)b 62 z!z17 (6)b

Values are expressed as a percentage of the value for basal membranes at term (mean f SEM). Number of measurements are shown in parentheses, “Value less than preterm, P
P
5. Basal membrane : microvillous membrane ratios for common protein kinase C (PKC) isoforms

PKC

isozyme I32

Termination (16-22 weeks)

Preterm (27-32 weeks)

Term

0.35 + 0.05 (7) 0.14 + 0.03 (5) 0.30 * 0.04 (11)

0.89 It 0.12” (3) 0.15 It 0.03 (4) 0.19 f 0.04 (3)

0.41 xt 0.09 (5) 0.35 + 0.11 (6) 0.47 + 0.14 (6)

(3841

weeks)

Ratios are given as mean + SEM; number of BM : MVM pairs from separate placental preparations is given in parentheses, “Value greater than term/termination. P~0.05.

cellular distribution and thus to elucidate the possible pathways by which PKC regulates cellular function. The syncytial nature of the placental epithelium does not permit isolation of syncytiotrophoblast cells from human placental tissue. Although there is an alternative route to obtain syncytiotrophoblast, through the culture of primary cytotrophoblast, the

(1996),

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degree to which these cells resemble their counterparts in vivo is questionable given the lack of exposure to the in utero environment. In the absence of syncytial cells, we have performed immunoblotting of syncytiotrophoblast plasma membranes purified from placental tissue in combination with immunocytochemisty of chorionic villous tissue to analyse the expression and distribution of PKC. The use of these experimental models raises an important question concerning the isoform profiles obtained for the plasma membranes and their relationship to total cellular content and distribution of PKC. Has there been any redistribution of PKC during the period between placental separation and formaldehyde fixation? The immunocytochemical data reported here shows clearly that there is little or no cytoplasmic staining in the syncytiotrophoblast using these anti-PKC antibodies, suggesting that the majority of the PKC in these cells is found bound to the plasma membranes, primarily the MVM. Recruitment of PKC to the plasma membranes might be ascribed to the effects of increased intracellular calcium and/or diacylglycerol following cellular breakdown. In most cases tissue fixation took place within 30 min of placental delivery, but in some cases tissue was fixed very rapidly after delivery. There was, however, no correlation between the duration of the pre-fixation period and the PKC membrane/ cytoplasmic partitioning profile. Moreover, endothelial and smooth muscle cells from the larger vessels and other nonsyncytial cells showed diffuse PKC staining of the cytoplasm under conditions where the syncytiotrophoblast showed little or no cytoplasmic staining. Many of the capillaries in these sections still contain erythrocytes, showing that these vessels were not completely washed out prior to or during fixation. Calcium concentrations are far greater in plasma than in the nominally calcium-free fixation medium, and therefore, one might expect the cells closest to the capillaries to be the first to demonstrate the effects of increased intracellular calcium on PKC membrane binding. The fact that these cells show cytoplasmic staining and the syncytiotrophoblast does not suggest that the localization of PKC in the syncytial cells is not an artefact of the preparation. The immunoblotting measurements were normalized to membrane protein because equal amounts of protein were loaded on to the gels. We have shown previously that the amount of protein per unit of phospholipid phosphorus differs between MVM and BM (Jansson, Wennergren and Illsley, 1993). There is an argument, therefore, for normalizing the PKC immunoblotting data to phospholipid phosphorus, which perhaps equates better to membrane surface area than membrane protein. Our prior data shows that the MVM contains -30 per cent more phospholipid phosphorus per unit membrane protein than the BM. Applying these figures to the data in Table 2, it is possible to see that while the absolute values of the BM : MVM ratio are altered, the asymmetry is still apparent. Mean values significantly less than unity for the BM : MVM ratios at term (Table 2) and standard errors averaging 26 per cent demonstrated that the asymmetry in PKC distribution is a consistent phenomenon, despite

Ruzycky,

Jansson

and Illsley:

Expression

of Protein

Kinase

C in Human

Placenta

probable phenotypic and genotypic heterogeneities in the placental tissue. The same data also shows that the degree of asymmetry is similar for the common isoforms, due possibly to a common mechanism producing this distribution. The asymmetry demonstrated by the unique localization at term of PKC a to the BM and PKC l31 to the MVM may reflect the differential distribution of PKC substrates between the opposing faces of the syncytial epithelium. The results of the immunocytochemistry experiments broadly support the data obtained by immunoblotting. The MVM displayed a high content of PKC isoforms compared with the BM, which showed little staining under these conditions. As observed in the immunoblotting studies, l32 and 6 were present consistently in greater quantity than the pl and y forms while the E isoform was undetectable. A note of caution should be appended, however, concerning the immunocytochemical data. Immunocytochemical experiments detected little PKC on the BM despite the presence of several isoforms detectable by immunoblotting. With respect to this difference, several points should be noted. First, the MVM is highly folded with an area approximately sixfold greater than that of the BM (Teasdale and Jean-Jacques, 1988). The convoluted structure of the MVM observed in the immunocytochemical sections presents a very high concentration of antigen compared with the BM and enhances the detection of microvillous PKC isoforms. Second, the nature of this procedure is such that antibody may recognize poorly or fail to recognize the antigenic site, which may not be accessible or in the appropriate antigenic conformation after tissue fixation. This may explain the inability to detect PKC E in the tissue sections. It should be noted that immunoblotting is an inherently more sensitive method than immunocytochemistry; data derived from immunocytochemical observations should, therefore, be interpreted cautiously and in the context of immunoblotting data. There is a dearth of information on the coupling of receptors to PKC responses in the placenta. There is evidence that PKC is involved in the signal transduction pathways for PTH (Alsat et al., 1993; Hellman et al., 1993), angiotensin II, ATP (Petit and Belisle, 1995; Vaillancourt, Petit and Belisle, 1995), follistatin (Shi, Zhang and Li, 1994) and HDL (Wu and Handwerger, 1992). We also have data to suggest that it plays a role in the endothelin pathway (unpublished data), however, there are currently no data linking individual isoforms to specific signalling pathways or functional systems in the syncytiotrophoblast. It is difficult to make a comparison with the previously presented information concerning placental PKC. The published reports noted the presence of CI and l3 isoforms but absence of the y isoform (Nomura et al., 1991; Tertrin-Clary et al., 1990; Tertrin-Clary, Chenut and de la Llosa, 1991). However, in light of the membrane/cytoplasm distribution observed here for the syncytial cells, it is probable that an analysis of the supernatant from whole placental homogenates would not reflect the syncytiotrophoblast PKC profile and might in fact be influenced significantly by the cytoplasmic

467

PKC profile from non-syncytial cells. A more recent report using similar sample material has confirmed the presence of the a and p isoforms (Bischof and Hammond, 1994). It should be noted that in this report we have not measured the distribution of all currently described PKC isoforms but rather for the major subset for which reliable antibodies for immunoblotting and immunocytochemistry are available. The presence and importance of the other isoforms mentioned (e.g. PKC n, h, u, 0) remains to be assessed. The immunoblotting data indicates that isoforms from each of the PKC classesare present on both MVM and BM. The distribution of these isoforms between the two membranes is asymmetric; for those isoforms which are common to the two membranes, the MVM showed a significantly higher level, a finding confirmed by the immunocytochemical data. There was also a differential distribution of the a and pl isoforms; the CY. isoform, when detected, was observed only on the BM while the pl isoform was associated only with the MVM. In the absence of information on substrate specificity, it is not possible to establish definitively the reasons for the observed differences in PKC distribution, however, the isoform distribution provides the potential for a novel mechanism whereby spatially selective activation of PKC can take place. The polarized distribution of moieties such as the insulin or /3-adrenergic receptors (Nelson, Smith and Jarrett, 1978; Whitsett, Johnson and Hawkins, 1979) provides a mechanism whereby intrasyncytial processes can be selectively activated by signals originating from mother or fetus. The existence of the three distinctive PKC classeson both MVM and BM provides another mechanism for the selective activation of specific syncytial processes by maternal or fetal signals. Agonistmediated stimulation of pathways that produce elevation of intracellular calcium, such as the IPs-coupled release of calcium from intracellular stores or the opening of calcium channels, will produce recruitment to and activation of cPKC isoforms on both MVMs and BMs. Release of diacylglycerol by pathways that do not affect intracellular calcium concentrations, such as hydrolysis of phosphatidylcholine (Billah and Canthes, 1990; Stabel and Parker, 1991), will be more selective, affecting only the nPKC class. Furthermore, there is the possibility of spatial selectivity in the activation of nPKC because (hydrophobic) diacylglycerol will for the most part be confined to the membrane in which it is generated. Thus a fetal agonist binding to the BM of the syncytiotrophoblast may cause release of diacylglycerol into the BM, activating only those processes on, or proximal to the BM; similar effects may result from the binding of an agonist from the maternal circulation to. the MVM. Apart from the asymmetry of the two opposing plasma membranes of the syncytiotrophoblast, the immunocytochemistry provided another interesting piece of datum, the observation that there was little or no PKC staining in the cytoplasm of the syncytial cells. This is in contrast to non-syncytial cells such as the smooth muscle cells in which there was diffuse cytoplasmic staining, under conditions in which no syncytial staining was observed. Neither membrane structure nor

468

Placenta

antigenic recognition are likely to be responsible for this difference because free PKC is clearly detectable in the non-syncytial cells. Thus in addition to the asymmetric epithelial distribution of the PKC isoforms identified in these membranes, the immunocytochemical data would suggest that the majority of syncytial PKC is plasma membrane-bound in vivo, and therefore, presumably active. The development of syncytial PKC through the second and third trimesters showed substantial changes. It is important to note first that the preterm placental tissue was taken from preterm deliveries which are, by definition, abnormal. Although these pregnancies were free from other complicating factors, the possibility exists that the (unknown) processes which led to preterm delivery also affected syncytial expression of PKC. There is however no other method by which preterm placental tissue can be obtained except for rare clinical situations, for reasons usually of maternal pathology. Moreover, it must be remembered that the individuals from whom these samples are taken are genotypically and phenotypically heterogeneous, even in the absence of other possible confounding factors such as errors in gestational age estimation. Analysis of the preterm data should, therefore, be made with these conditions in mind. The u. isoform was not consistently detectable in the termination and preterm periods, and when present, was observed at extremely low levels. It is clear that the changes in PKC over gestation are not a generalized effect on PKC or protein expression; in MVM, the p2 and E levels rose over the course of gestation while the l31, y and [ isoforms showed no significant changes. In the BM, PKC a and y were essentially undetectable before term while the p2, I and c isoforms on the BM showed significant changes over the 16-40 week period. The asymmetry in PKC expression between the MVM and BM was observed throughout gestation with the -MVM maintaining a consistently higher content of the common isoforms. Developmental changes in PKC isoform expression have also been observed in other tissues (Jiang et al., 1992; Saxena et al., 1994). In the absence of isoform substrate specificities it is difficult to determine the functional rationale for the gestational changes in PKC isoform expression. The fetus (and placenta) passesthrough several major developmen-

(1996),

Vol. 17

tal stages from 8840 weeks of gestation; the initial stages involve structural organization, organogenesis and body growth followed later by a phase characterized primarily by substantial weight gain and finally a period of maturation in preparation for birth. A huge variety of hormonal changes occur during these periods, many of which are likely to involve PKC modulation; the gestational changes in PKC expression and distribution are likely part of the rapidly altering endocrine environment responsible for fetal development. The syncytiotrophoblast contains an isoform (0 which may be constitutively active because it requires neither calcium nor diacylglycerol for activation (Kochs et al., 1993; Nakanishi and Extort, 1992). Despite this, there is some uncertainty concerning potential activation factors. PS and other acidic phospholipids appear to activate PKC c although this may involve potentiation of the effects of other agents such as arachidonic acid and other free fatty acids. PKC < is also activated by another class of phospholipids, the inositol phospholipids (Nakanishi et al., 1993), primarily phosphatidylinositol-3,4,5triphosphate (PIP,), but also possibly by the diphosphate derivatives (Kochs et al., 1993; Nakanishi et al., 1993). PIP, is the product of phosphatidylinositol 3-kinase activity, an enzyme that is activated via binding to activated growth factor receptors, suggesting another route for PKC activation. In fact Sung and Goldfine have suggested that phosphatidylinositol 3-kinase should be regarded as a non-tyrosine phosphorylated member of the insulin receptor signalling complex (Sung and Goldfine, 1992), providing a route for activation of PKC separate from the classical G-protein/phospholipase C mediated pathways. Activation of PKC has also been observed via direct interaction of the insulin receptor with PKC a in CHO cells (Liu and Roth, 1994), further supporting the existence of alternate pathways for PKC activation. The combination of substrate specificities, alternate routes for activation and the possibility of spatially separate maternal and fetal activation pathways provides a means for the selective and specific modulation of the phosphorylation state of a wide range of proteins via PKC, giving it an extremely important role in syncytiotrophoblast regulation.

ACKNOWLEDGEMENTS The authors would like to thank the staff of the Birth Center at the University of California Medical tissue. The research was supported in part by NIH grant ROl HD23498 to N.P.I. and a travel grant

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