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Published ahead of print on October 9, 2003, doi:10.1165/rcmb.2003-0266OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 510-518, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0266OC

Mesothelial Differentiation as Reflected by Differential Gene Expression

Xiaojuan Sun*, Miklós Gulyás* and Anders Hjerpe

Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden

Address correspondence to: Miklós Gulyás, M.D., Ph.D., Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, F46 Huddinge University Hospital, SE-14186 Stockholm, Sweden. E-mail: miklos.gulyas{at}labmed.ki.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human mesothelial cells obtained from benign effusions retain their proliferative capacity and grow uniformly either with a fibroblastic or epithelioid morphology in vitro. These cultures therefore provide a model for the process of mesothelial differentiation in vivo. To study this differentiation, we isolated differentially expressed genes obtained by suppression subtractive hybridization. Of the nine genes found to be overexpressed in fibroblastic mesothelial cells, three are matrix-associated (integrin {alpha}5, collagen binding protein 2, human cartilage glycoprotein 39), whereas the others are associated with a proliferative cell type (14–3-3 {epsilon}, plexin B2, N33, and three genes encoding ribosomal elements). Seven of the eight genes upregulated in the epithelioid phenotype are related rather to specialized functions, such as metabolism (aldose reductase, lecithin:cholesterol acyltransferase, ATPase 6), cytoskeletal composition (cytokeratins 7 and 8), and regulation of differentiation (granulin, annexin II). Immunohistochemistry with available antibodies to six of the differentially expressed gene products confirmed the differences also in pleural tissues, where submesothelial cells displayed the fibroblastic markers, whereas surface cells displayed the epithelioid markers. In summary, this approach revealed a pattern of genes coordinately regulated during mesothelial differentiation and suggests that mesothelium may regenerate also by recruiting cells from the submesothelial layer. Some of the gene products may also be useful markers for differentiation and activation in serosal tissues.

Abbreviations: aldose reductase, AR • collagen binding protein 2, CBP2 • cytokeratin 7, CK7 • cytokeratin 8, CK8 • digoxigenin, DIG • glyceraldehyde-3-phosphate dehydrogenase, G3PDH • human cartilage glycoprotein 39, HCgp39 • protein kinase C, PKC • suppression subtractive hybridization, SSH • Wilms' tumor susceptibility gene 1, WT1


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mesothelial cells covering the serous membranes are unique in the sense that they are of mesodermal origin (1), but retain their ability to differentiate toward an epithelioid morphology throughout adult life. The cells obtained from the serosal surface can be cultured. Within a collagen matrix they take on a spindle shape and then migrate toward the surface (2). On reaching the surface, they change their morphology and spread to cover it with polygonally-shaped epithelioid cells. This process resembles embryonic nephrogenesis and formation of the surface (celomic) epithelium of the ovaries. During the transition of mesenchymal cells into cells having an epithelial growth pattern, these three tissues (kidney, ovary, mesothelium) express the Wilms' tumor susceptibility gene 1 (WT1). In contrast to the transient embryonic expression during renal epithelialization, continuous expression of WT1 during adult life has been described only in gonadal and mesothelial cells (3, 4), implying the preserved transdifferentiational characteristics of these cells. Unlike the mesothelium, these other mesenchymal–epithelial transitions (kidney, ovary) result in tissues with adhesion mechanisms and ultrastructure of epithelial type. The surface mesothelium acquires only some of these epithelial characteristics, such as polygonal cell shape and the ability to form cytokeratin intermediate filaments (5, 6), desmosomes, and a basement membrane (6, 7); but others, such as E-cadherin adhesion (8), glycocalyx, and microvillus-associated core rootlets (9), are less typical or absent.

In case of injury, the mesothelium may regenerate via two mechanisms, which differ in principle. Surface mesothelial cells have retained their capacity to proliferate and, after exfoliation or migration, they may rapidly cover the defect. This is supposed to be the main regeneratory mechanism, particularly if the basement membrane is intact. Following such an injury, the polygonal epithelial-like cells temporarily transform into a spindle-shaped fibroblastic morphology. These cells attach or migrate to the defect, where they later become flattened to cover the surface (10, 11). Mesothelial regeneration has also been assumed to occur from fibroblast-like multipotential subserosal cells. Particularly when the basement membrane is affected, these cells are supposed to migrate to the surface, during which process they gradually acquire epithelial characteristics (6, 12, 13). A similar variability in growth phenotype is also seen in malignant mesothelioma with fibroblastic and/or epithelioid morphology. In these tumors, the fibrous growth pattern is associated with a more aggressive behavior (14).

In various benign disorders, such as circulatory insufficiency, liver cirrhosis, or serosal inflammation, an effusion is present that sometimes contains many cells from proliferative or regenerative mesothelium. Both the spindle-shaped and polygonal cell morphologies can be induced by cultivating these exfoliated cells—i.e., the short-term cultures consistently show either a fibroblast-like or epithelioid growth pattern (Figure 1). These patterns are stably retained under standard conditions throughout subsequent passages (15). The fibroblastic morphology obtained mimic the in vivo growth pattern of reactive subserosal spindle-shaped cells (6, 12, 16), whereas the cells in epithelioid cultures resemble those covering the serous surface. These fibroblastic and epithelioid benign cultures thus allow the study of the mechanisms on which mesothelial differentiation depends.



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  		Figure 1. Mesothelial cells from benign effusions grow uniformly with fibroblastic (left) or epithelioid (right) morphology in culture; scale bar, 25 µm.

 
Suppression subtractive hybridization (SSH) permits examination of differential gene expression in the stages of differentiation and in various conditions of growth (17). Other techniques, such as DNA microarray, may detect larger numbers of differentially expressed genes; on the other hand, the differentially expressed genes obtained by SSH represent the largest differences, and the number of genes acquired is sufficient to discern patterns of gene expression. SSH has thus been used to study malignant mesothelioma cell sublines having various morphologic phenotypes (18). Although several factors, such as the WT1 and proteoglycans, are associated with mesothelial differentiation (15, 19, 20), the genes underlying the mechanism that regulates this transformation into an epithelioid or fibroblastic phenotype are not completely understood. To obtain insight into how benign mesothelial cells differentiate, we have characterized differentially expressed genes by SSH, comparing pooled cDNAs from four epithelial-like and four fibroblastic short-term cultures. Amplimers from such genes were cloned and sequenced to determine the origin of the gene, the specificity of the analysis being verified by Southern blot and RNA dot blot. To confirm the findings in vivo, we also studied benign pleural mesothelium immunohistochemically with commercially available antibodies to differentially expressed gene products.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures
Nonmalignant pleural and ascitic effusions were centrifuged and the cells seeded in 75 cm2 tissue culture flasks containing RPMI-1640 medium supplemented with 5% fetal bovine serum, 5% calf serum, and antibiotics (penicillin 100 µg/ml, streptomycin 100 µg/ml, and gentamicin 50 µg/ml) in humidified 5% (vol/vol) CO2 at 37°C. The culture medium was changed every other day. All cell cultures included in the study grew uniformly with either morphologic phenotype: fibroblast-like or epithelioid (Figure 1). These growth patterns are stable during several passages, and for the present analysis the cultures were harvested after 1–2 passages at a cell confluence of ~ 80%. The mesothelial origin of the cells was shown as previously (15). Briefly, the cells express the WT1 gene and they show immunocytochemical reactivity for broad-range cytokeratins, vimentin, {alpha}-smooth muscle actin, and calretinin. Electron microscopy shows varying densities of surface microvilli. The fibroblastic cell cultures were somewhat more proliferative, as reflected by ~ 30% less time to reach confluence. The study was approved by the Institutional Ethical Committee of Huddinge University Hospital/Karolinska Institutet.

Preparation of Total RNA and Poly (A+) RNA
Total RNA was isolated from eight short-term cultures—i.e., four with epithelial and four with fibroblastic growth patterns, using the High Pure RNA Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany). Poly (A+) RNA was purified from this total RNA, using the Oligotex mRNA Midi kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions.

Suppression Subtractive Hybridization
The analyses were done in triplicate, using double-stranded cDNA synthesized from 2 µg of poly (A+) RNA. This RNA was obtained by pooling preparations from short-term cultures of similar phenotype. For both epithelioid and fibroblast-like cells, four cultures were sufficient to obtain the desired amounts. Each of the two pools obtained were assayed as tester against the other one, using the Clontech PCR-select cDNA subtraction kit (Clontech, Palo Alto, CA), as described elsewhere (18).

First- and second-strand cDNA synthesis and blunt-ending of this DNA were then performed with T4 DNA polymerase. Restriction enzyme digestion was performed with 15 U RsaI for 2 h at 37°C. The resulting cDNA fragments were then purified and ligated with T4 DNA ligase to give one preparation labeled with adaptor 1 and one with adaptor 2. Double-stranded driver cDNA was similarly prepared, without adding adaptor sequences.

To each of two tubes containing adaptor 1– or adaptor 2–ligated tester cDNA, the driver cDNA was added in 23-fold excess. The samples were mixed and incubated in a thermal cycler (Gene AmpR PCR System 9600; Perkin-Elmer, Norwalk, CT), and denaturation obtained at 98°C for 1.5 min. This was followed by annealing at 68°C for 12 h. After mixing hybridized testers 1 or 2 with additional freshly-denatured driver cDNA, the mixture was kept overnight for a second hybridization at 68°C. Each subtracted tester preparation was then analyzed by a nested polymerase chain reaction (PCR) amplification and subsequent electrophoresis in 2% agarose gel.

Cloning and Sequencing of the Obtained Amplimers
The bands obtained from electrophoresis of the nested PCR products were excised and purified by the QIAEX II agarose gel extraction kit (Qiagen GmbH). The isolated amplimers were then digested with RsaI and cloned, using the pGEM- T Easy Vector System II (Promega Corp., Madison, WI, USA). Double-stranded plasmid was prepared using the QIA filter plasmid kit (Qiagen GmbH) and sequenced with an ABI 377 sequencer (Applied Biosystems Division, Foster City, CA, USA), the analysis being performed by CyberGene AB (Novum Research Park, Karolinska Institutet, Huddinge, Sweden).

Southern Blot Analysis
The differential expression of all phenotype-specific transcripts was confirmed by Southern blot analysis of the nested PCR amplimers. Digoxigenin (DIG)-labeled probes (25 bp) were designed to recognize specific sequences in the cDNA clones obtained. Amplified subtracted tester cDNA (300 ng/lane) were run on a 1.5% agarose gel and transferred to a Sure Blot Hybridization membrane (Intergen Co., Purchase, NY), using a vacuum transfer apparatus. The membranes were baked at 80°C for 2 h, and hybridization performed in accordance with the manufacturer's protocol (Sure Blot Chemi Hybridization and Detection Kit Procedure; Intergen Co.). To evaluate subtraction efficiency, PCR-amplified unsubtracted and subtracted cDNAs were compared after electrophoresis on a 1.5% agarose gel. The membranes were hybridized with DIG-labeled probes for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and for aldose reductase and integrin {alpha}5; these latter two sequences found to be differentially expressed by the respective phenotype. All these experiments were performed in triplicate.

RNA Dot Blot Analysis
Aliquots from the pooled fibroblastic and epithelioid mRNAs were diluted to give final concentrations of 250, 50, and 10 ng/µl. Following denaturation, 2 µl of each aliquot were spotted on Sure Blot Hybridization membranes. After blocking reactions (DIG easy Hyb; Roche), the hybridization with the DIG-labeled probes was performed overnight at 50°C. Membranes were then washed, blocked, and incubated with an anti-DIG alkaline phosphatase–conjugated antibody (Roche). Hybrids were visualized by developing with chemoluminescent alkaline phosphatase technique (Ready-to-use-CSPD; Roche). The same blots were re-probed with a DIG-labeled 18S RNA probe control to ensure sufficient loading of RNA (21). The analyses were done three times.

Immunohistochemistry
Immunohistochemical reactions in native pleural tissues were performed on formalin-fixed paraffin-embedded sections. The specimens with nonmalignant pleural disorders were retrieved from the archives in the Laboratory for Clinical Pathology and Cytology, Huddinge University Hospital (Stockholm, Sweden). Three specimens contained quiescent mesothelium and three represented mesothelial tissues with signs of reactive processes and regeneration.

After antigen retrieval in 10 mM sodium citrate buffer, pH 6.0 at 95°C for 5 min, and subsequent cooling for 20 min, endogenous peroxidase activity was abolished by 0.6% H2O2 in methanol and nonspecific binding was blocked with 3% normal rabbit serum. Incubation with specific antibodies to the signal transducer protein 14–3-3 {epsilon} (Chemicon International, Inc., Temecula, CA), integrin {alpha}5 (Chemicon International), collagen-binding protein 2 (Anti-Hsp47; Stressgen Biotechnologies Corp., Victoria, BC, Canada), annexin II (Zymed Laboratories, Inc., San Francisco, CA), cytokeratin 7 (DAKO Corp., Carpinteria, CA) and cytokeratin 8 (DAKO Corp.) was followed by biotin-conjugated secondary antibody (Multy-Link for 14–3-3 {epsilon} and integrin {alpha}5, and AB2 for the remaining 4 primaries; DAKO, Copenhagen, Denmark), both done for 30 min at room temperature. The streptavidin-biotin-peroxidase method with diaminobenzidine as the substrate-chromogen was used for visualization (ChemMate Detection Kit; DAKO).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Southern blot showed that the subtraction eliminated all detectable expression of housekeeping genes, while differentially expressed genes were readily detected after subtraction and secondary PCR amplification (Figure 2). In total, 30 cloned amplimers were sequenced, and the results were compared with the GenBank database, using the BLAST program (22). The sequencing of the amplimers obtained showed several cDNA inserts with the same sequences, probably as an effect of large amounts of mRNA. We found 17 different cDNA sequences, ranging in size from 200–500 bp (Table 1). Eight of these genes were differentially expressed in the epithelioid cells, whereas the remaining nine genes were obtained with fibroblastic cDNA as tester. Comparison of the sequences obtained with the database sequences indicated that they all came from previously identified human genes.



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  		Figure 2. The efficiency of SSH is shown with Southern blot. Hybridization for G3PDH (left membrane) gives two bands in the unsubtracted cDNA preparation (epithelioid cells in lane 1, fibroblastic cDNA gives similar pattern [not shown]). These bands disappear after subtraction and subsequent amplification (cDNA from epithelioid cells as tester in lane 2 and from fibroblastic cells in lane 3). Hybridization of a parallel membrane (right) with probes for aldose reductase and integrin {alpha}5 shows no reactivity in unsubtracted cDNA (lane 4), whereas the subtracted and amplified preparations show distinct bands for aldose reductase in epithelioid tester cDNA (lane 5) and for integrin {alpha}5 in fibroblastic tester cDNA (lane 6).

 

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  		TABLE 1 Genes differentially expressed in fibroblastic and epithelioid benign mesothelial cells

 
The Southern blot (Figure 3) analysis also showed that the cloned cDNAs were differentially expressed and amplified only in the preparation from which the clone had been obtained. The genes detected in the fibroblastic cells were as follows: ribosomal 28S rRNA, acidic ribosomal phosphoprotein P0, ribosomal protein L19, 14–3-3 {epsilon} protein, plexin B2, N33, integrin {alpha}5, collagen binding protein 2 (CBP2), and human cartilage glycoprotein 39 (HCgp39). Similarly, granulin, annexin II, cytokeratin 7 (CK7), cytokeratin 8 (CK8), aldose reductase, lecithin:cholesterol acyltransferase, mitochondrial ATPase 6, and nuclear pore complex-associated protein TPR were met in the epithelioid cells. Accordingly, the RNA dot blot analysis (Figure 4) confirmed the differential gene expression. mRNAs for most of these genes were detected in the respective phenotype only, except HCgp39, which was shown in both, although with different amounts.



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  		Figure 3. Southern blot analysis of the cDNA after SSH. Expressions of collagen binding protein 2 (CBP2), human cartilage glycoprotein 39 (HCgp39), 14–3-3 {epsilon}, and integrin {alpha}5 were detected only in subtracted cDNAs from fibroblastic (fb) cells (upper panel); whereas CK7, aldose reductase (AR), granulin, CK8, and nuclear pore complex–associated protein TPR (NPCAP) were found in subtracted cDNAs from epithelioid (ep) cells (lower panel).

 


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  		Figure 4. RNA dot blots (500, 100, and 20 ng mRNA) of differentially expressed genes. Human cartilage glycoprotein 39 (HCgp39), CBP2, and integrin {alpha}5 were largely expressed in fibroblastic (fb) cells, whereas CK7, granulin, AR, and CK8 were detected in epithelioid (ep) cells. 18S RNA was probed for control.

 
Attempts to cluster the genes obtained as differentially expressed by the mesothelial cultures indicate distinct differences between the two phenotypes (Table 1). Genes overexpressed by cells with fibroblastic morphology can be related to proliferation (six genes, three of them encoding ribosomal elements) or cell–matrix interaction (three genes). The genes correspondingly upregulated in the epithelioid phenotype encode proteins that have either been directly associated with tissue differentiation (two regulatory genes), cytoskeleton (two genes), or enzymes involved in metabolism (three genes). Only one gene obtained from the latter phenotype did not fit into any of these groups.

Immunohistochemistry with the available antibodies to six of the gene products on reactive pleural tissues confirmed in vivo the differential gene expression obtained by SSH. In tissue areas showing mild features of activation (thickened and cellular submesothelial tissue, slightly increased height of surface cells; Figure 5), only the submesothelial fibroblastic cells gave distinct reactivities for 14–3-3 {epsilon} protein, integrin {alpha}5, and CBP2, whereas the surface mesothethelial cells remained mostly unreactive (Figures 5A–5C). In these sites, immunoreactivities for epitopes of genes upregulated in the epithelioid phenotype (annexin II, CK7, and CK8) yielded an opposite staining pattern, labeling only the mesothelial cells on the serosal surface (Figures 5D–5F). In mesothelial areas with marked cytomorphologic signs of activation (serosal surface covered with cuboidal cells), both epithelioid and fibroblastic cells showed intermediate staining characteristics with coexpression of both these "epithelial" and "fibroblastic" markers. On the other hand, in quiescent mesothelium the "fibroblastic" markers were almost nonreactive in both the submesothelial and surface cells, whereas the "epithelial" markers labeled only the lining mesothelial cells.



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  		Figure 5. Mildly reactive mesothelial tissue. Beneath the flat or slightly thickened lining cells the subserosal fibroblast-like cells are increased in number and give distinct immunoreactivities for 14–3-3 {epsilon} protein (A), integrin {alpha}5 (B), and CBP2 (C). In contrast, annexin II (D), CK7 (E), and CK8 (F) give an opposite pattern with strong staining only in the superficial mesothelial cells. Surface mesothelial cells are indicated with arrows. (Immunoperoxidase-DAB, counterstained with hematoxylin; scale bar, 50 µm.)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Differentially Expressed Genes
The genes for ribosomal 28S rRNA, acidic ribosomal phosphoprotein P0, and ribosomal protein L19 were all highly expressed in the fibroblastic morphology. The rate of rRNA transcription directly affects ribosome production and protein synthesis and, in proliferating immature myeloid cells, it is higher than in the corresponding cells after their differentiation (23). An increase in the expression for acidic ribosomal phosphoprotein P0 (24) and ribosomal protein L19 (25) has been found in carcinomas (liver, colon, breast), and this has been associated with a more malignant potential. This upregulation of ribosomal genes may therefore be related to an increase in proliferation, presumably as part of the protein synthesis needed by dividing cells.

14–3-3 {epsilon}, a protein isoform involved in signal transduction, has been implicated in the activation of protein kinase C (PKC; 26, 27), and it is therefore correlated to cell division (28). During nephrogenesis, the mRNA level of this gene is high in proliferating early mesenchyme, and it declines markedly as the tissue differentiates and early nephrogenic condensates epithelialize (26). Our findings of an upregulated 14–3-3 {epsilon} gene in the fibroblastic cultures and presence of the epitope in the submesothelial fibroblasts at reactive tissue areas (Figure 5A) support the view that these cells are less differentiated and more proliferative in nature.

Plexin B2 (KIAA0315) is a transmembrane receptor belonging to the large family of plexins. These proteins control cell dissociation, and may in this way facilitate cell proliferation in a variety of tissue (29). Their regulatory proteins, the semaphorins, have been found to be overexpressed in metastatic cancer cell lines, unlike the nonmetastatic parental cells (30). Expression of plexin B2 mRNA is also correlated to the proliferative capacity of brain tumors, undifferentiated glioblastomas having a much higher expression level than low grade astrocytomas (31).

N33, otherwise known as putative prostate cancer tumor suppressor, is a gene involved in the regulation of cell proliferation, although its function remains to be precisely clarified. The expression of this gene can be lost in malignant tumors (32). In mature benign tissues N33 expression has been shown to decline with age, a change correlated with an increase in the risk of carcinogenesis (33). The finding that N33 and other proliferation-associated genes are differentially expressed in the fibroblastic mesothelial cultures is interesting.

Integrin {alpha}5, the constant subunit of the fibronectin receptor, participates in cell–matrix interactions. Its activation by fibronectin promotes cellular migration and proliferation (34). When integrin {alpha}5 expression was enhanced stimulating vascular smooth muscle cells with cytokines, it resulted in a proliferating phenotype with a spindle-shaped morphology (35). In transfection experiments, the overexpression of this integrin subunit resulted in myoblast proliferation, but that of the {alpha}6 subunit promoted differentiation (34). In our mesothelial model, the increased expression of integrin {alpha}5 in the fibroblastic cells also suggests that this phenotype is less differentiated. This was also confirmed by the distinctive immunoreactivity in submesothelial fibroblasts of activated pleural tissues (Figure 5B).

Collagen-binding protein 2 (CBP2; also known as Colligin 2 or HSP47) is a heat shock protein that acts as a molecular chaperon in the biosynthesis of collagens (36). An increase in the synthesis of this glycoprotein occurs in fibrous disorders of the kidney (37) and the peritoneum (38). The immunoreactivity of the pleural tissue for this protein was mainly confined to the submesothelial fibroblast-like cells of reactive areas (Figure 5C), as also shown in the peritoneum (38). This indicates that the mesothelial cultures with spindle-shaped phenotype are truly fibroblastic, CBP2 being a component of the collagen and matrix-producing machinery.

Human cartilage glycoprotein 39 (HCgp39; also known as YKL-40) is a 40-kD protein previously found in chondrocytes and synovial fibroblasts (39). Through its heparin-binding properties, HCgp39 is believed to play a role in adhesion of cells to the extracellular matrix and also in cell migration (40). It is associated with fibroblast activation, and high levels are seen during an increase in the turnover of these cells (39, 41). Thus, in liver tissue, slight immunoreactivity to HCgp39 occurs in the portal area, which increases during active fibrogenesis (40). The upregulation of the HCgp39 gene in the fibroblastic mesothelial phenotype seems to reflect the mesenchymal nature and active cell–matrix interaction of these cells.

The granulins or epithelins constitute a family of ~ 6 kD cysteine-rich growth modulatory peptides which are generated from a common precursor (42–44). Although the common granulin sequence is detectable in most tissues, the expression is greatest in epithelial ones (44). High mRNA levels are found especially in young epithelial cells with mitotic activity, whereas terminally differentiated cells show low expression levels (45). Of the seven known forms of granulin, only granulin A (epithelin 1) and granulin B (epithelin 2) have been assigned a biological activity: the former promotes proliferation in epithelial cells, and the latter has a contrary effect (43). Thus, the ratio of specific granulin/epithelin molecules seems to be important in orchestrating epithelial differentiation from an early to a terminal phase. The granulin mRNA found differentially expressed in the epithelioid mesothelial phenotype represents a common sequence, and provides no information on the granulin subtypes. Nevertheless, this finding supports the concept that the increased expression of granulins plays a role in promoting epithelial differentiation (46). This also harmonizes with the view that the epithelioid mesothelial phenotype is not terminally differentiated, and represents an active phase of epithelial differentiation, in which process this phenotype is preceded by the fibroblastic one.

Annexin II (also called lipocortin II or p36) belongs to the large family of annexins, a group of calcium and phospholipid binding proteins that reversibly bind membranes. Annexin II, as a cell membrane–organizing compound, has been implicated in membrane trafficking and suggested as a regulator of cellular differentiation (47). It seems to inhibit PKC activity, presumably by regulating the various PKC isoforms (48). An increase in the expression of annexin II corresponds to differentiation rather than to proliferation in both normal liver development (49) and malignant tumors (48, 50–52). In pleural tissue, the superficial mesothelial cells showed marked immunoreactivity for annexin II (Figure 5D; 53), which is in line with the increase in mRNA expression for this gene in the epithelioid phenotype.

Cytokeratins (CKs) are commonly used as markers of epithelial differentiation. A simultaneous immunocytochemical reactivity for vimentin and a wider spectrum of CKs are regarded as characteristic of mesothelial lineage (54, 55). CK7 and CK8, known to label simple, one-layered epithelia specifically (56), induce marked immunoreactivity in surface mesothelial cells, but the submesothelial fibroblast-like cells, which stain with antibodies for a broad spectrum of CKs (13), are not reactive (Figures 5E and 5F). The differential expression of these CK genes and the resulting abundance in CK7 and CK8 intermediate filaments are therefore related to the epithelioid phenotype of the mesothelium, indicating a more advanced stage of differentiation.

Aldose reductase (AR) is an NADPH-dependent enzyme that catalyzes the reduction of glucose to sorbitol. Marked expression of AR has been described in many differentiated tissues, such as lung, liver, and brain (57). It is abundantly present in the renal tubuli (58). In the newborn rat kidney, AR mRNA expression increases rapidly during maturation (59) as part of the developmental program (60). The expression of AR may thus be involved in the epithelialization of mesenchymal precursor cells and the upregulation of this gene in the epithelioid cells of the mesothelium is therefore associated with a more advanced stage of differentiation in this tissue also.

Lecithin:cholesterol acyltransferase (LCAT) is an enzyme that mediates the formation of cholesteryl esters, whereas ATPase 6 is a mitochondrial enzyme catalyzing ATP production (61). A substantial production of lecithin has been demonstrated in lining mesothelial cells, suggesting that this phospholipid participates in reducing the friction between the opposing serosal surfaces (62). The high expression of these genes involved in lipid and energy metabolism in the epithelial-like mesothelial phenotype seems to be a functional marker of differentiated cells related to a more metabolically active state.

Nuclear pore complex–associated protein TPR (also called as p270/Tpr) is a 270-kD polypeptide that is a ubiquitous component of the intranuclear filaments attached to the nuclear pore complex. As such a protein, it may be involved in filament-guided nucleo-cytoplasmic transport processes (63). It is more abundantly expressed in the epithelioid mesothelial cells, which suggests enhanced communication between nucleus and cytoplasm; however, the full function of this protein in association with differentiation remains to be elucidated.

General Discussion
Many genes are supposed to be involved in the regulation of mesothelial differentiation. With the present protocol for SSH, only marked differences in expression of genes (> 20-fold) give suitable amplimers. Thus the present analysis yields a random sample of the numerous differentially expressed genes that can be detected by microarray analysis (64). Still, the sample of the 17 genes obtained shows some distinctive patterns, the limited number of genes making this functional clustering easier. Of the genes overexpressed in fibrous cells, some are matrix-associated and therefore related to a mesenchymal phenotype, although most are associated rather with a less differentiated cell, which has marked features of proliferation. The genes upregulated in epithelioid mesothelial cells are associated instead with specialized functions, such as metabolism, cytoskeletal composition, and regulation of a more differentiated cell type.

The exfoliated cells that result in cultures with fibroblastic morphology may theoretically originate from fibroblast-like subserosal cells after injury to the tissue. However, this growth pattern was the commonest one in cultures from transudates,—i.e., effusions not associated with destruction of the basement membrane. It therefore seems more likely that all the cultured cells of both morphologies were derived from exfoliated superficial serosal cells that in vivo have an epithelioid phenotype. These cells can obviously grow with either of the two morphologies—i.e., they can transdifferentiate to a fibroblastic phenotype. The pattern of growth during cultivation may well depend on stimuli before the shedding of cells from the pleural or peritoneal surface. This view is supported by the finding that epithelioid differentiation was mainly obtained with cells from acute inflammatory exudates (15), whereas all fibroblastic cultures came from effusions containing numerous mesothelial cells with cytomorphologic signs of activation (enlargement, vacuolation, variations in size, increase in the nuclear-cytoplasmic ratio, chromatin irregularities, binucleation), so-called "mesotheliosis."

A similar transdifferentiation of epithelioid mesothelial cells into mesenchymal phenotype has been described in cultures from effluents obtained during continuous ambulatory peritoneal dialysis (65). Transforming growth factor ß1 (TGF-ß1) and interleukin-1ß, detected in these effluents, have been ascribed a role in this process (64–66). Conversion to fibroblastic morphology can also be achieved by adding epidermal growth factor (EGF; 67). The type of mesothelial cell growth in vitro thus does not seem to be a chance phenomenon; the precise mechanism behind this transdifferentiation, however, remains to be elucidated.

Available antibodies allow immunohistochemical studies of proteins encoded by three of the genes upregulated in the more proliferative fibroblastic phenotype (14–3-3 {epsilon}, integrin {alpha}5 and CBP2) in vivo. In resting mesothelium, these markers gave almost no reactivity in both surface and submesothelial cells. In mesothelial areas showing features of mild activation, the reactivities for the above markers were confined mainly to fibroblastic subserosal cells, while the immunoreactivities in the flat superficial cells were faint or absent (Figures 5A–5C). However, some reactivity may also occur in the superficial epithelial-like mesothelial cells, particularly in areas where these surface cells have become cuboidal in form as a sign of marked activation. In these areas, the subserosal fibroblastic cells are also increased in numbers and give an even stronger reactivity with these markers. The available immunohistochemical markers for upregulated genes related to epithelioid differentiation (annexin II, CK7, CK8) gave strong reactivity almost exclusively in surface mesothelial cells of quiescent and mildly activated serosal areas, where these cells were flat or slightly thickened (Figures 5D–5F). In areas with marked signs of reactive processes and regeneration (cuboid superficial cells and occasional stratification with formation of papillary projections; 16), some reactivity for these "epithelial" markers was also seen in subserosal fibroblastic cells,—i.e., they showed an intermediate immunophenotype with both groups of markers simultaneously present.

The findings indicate that mesothelial cells have the capacity to shift between epithelioid and fibrous growth patterns, as previously also suggested by Bittinger and coworkers (2) and Mutsaers (68), and both of these cell phenotypes may therefore contribute to the process of regeneration. This is particularly obvious when there is a marked regenerative or proliferative stimulus of the tissue. In these situations, both kinds of cells present an immunophenotype, intermediate to that found in less activated cells of fibrous and epithelioid morphology. Reparative processes may thus recruit mesothelial cells from two sources: from surface mesothelial cells (10) and, in case of a more severe injury, from subserosal fibroblastic cells. That the latter act as a progenitor population was previously based on coexpression of vimentin and cytokeratins seen in these cells at sites of active serositis and serosal injury (6, 12). Our finding of the overlapping immunophenotypes with additional epitopes further supports this view.

Taken together, the SSH revealed a limited number of genes coordinately regulated during the differentiation of mesothelial cells. The differential gene expression in the fibroblast-like mesothelial cultures corresponds best to the immunophenotype of activated subserosal fibroblastic cells in vivo. Although the epithelioid and fibroblastic mesothelial cells are closely related, the fibrous ones are more proliferative and represent a less differentiated phenotype. Therefore the transition of surface mesothelial cells into cells with fibroblastic morphology indicates a dedifferentiation, whereas the opposite transformation corresponds to a process of development toward a specialized cell type. This concept may explain why the sarcomatous variant of malignant mesothelioma behaves more aggressively, also suggesting that some of the differentially expressed gene products may be useful markers for differentiation and activation in both benign and malignant serosal tissues.


   Acknowledgments
 
This work was supported by the Swedish Cancer Fund (grant no. 2485) and the Swedish Heart and Lung Fund.


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form July 17, 2003

Received in final form October 7, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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