Journal of Histochemistry and Cytochemistry, Vol. 49, 1143-1154, September 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Prolyl 4-hydroxylase Isoenzymes I and II Have Different Expression Patterns in Several Human Tissues

Ritva Nissia, Helena Autio–Harmainenb, Pia Marttilaa, Raija Sormunenb, and Kari I. Kivirikkoa
a Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu
b Department of Pathology, University of Oulu, Oulu, Finland

Correspondence to: Kari I. Kivirikko, Collagen Research Unit, Biocenter and Dept. of Medical Biochemistry, University of Oulu, PO Box 5000, FIN 90014 Oulu, Finland. E-mail: kari.kivirikko@oulu.fi


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Prolyl 4-hydroxylase plays a central role in the synthesis of all collagens. We have previously reported that the recently identified Type II isoenzyme is its main form in chondrocytes and possibly in capillary endothelial cells, while Type I is the main form in many other cell types. We report here that the Type II isoenzyme is clearly the main form in capillary endothelial cells and also in cultured umbilical vein endothelial cells, whereas no Type I isoenzyme could be detected in these cells by immunostaining or Western blotting. The Type II isoenzyme was also the main form in cells of the developing glomeruli in the fetal kidney and tubular structures of collecting duct caliber in both fetal and adult kidney, in occasional sinusoidal structures and epithelia of the bile ducts in the liver, and in some cells of the decidual membrane that probably represented invasive cytotrophoblasts in the placenta. Osteoblasts in a fetal calvaria, i.e., a bone developing by intramembranous ossification, stained strongly for both types of isoenzyme. The Type I isoenzyme was the main form in undifferentiated interstitial mesenchymal cells of the developing kidney, for example, and in fibroblasts and fibroblastic cells in many tissues. Skeletal myocytes and smooth muscle cells appeared to have the Type I isoenzyme as their only prolyl 4-hydroxylase form. Hepatocytes expressed small amounts of the Type I enzyme and very little if any Type II, the Type I expression being increased in malignant hepatocytes and cultured hepatoblastoma cells. The data suggest that the Type I isoenzyme is expressed especially by cells of mesenchymal origin and in developing and malignant tissues, whereas the Type II isoenzyme is expressed, in addition to chondrocytes and osteoblasts, by more differentiated cells, such as endothelial cells and cells of epithelial structures. (J Histochem Cytochem 49:1143–1153, 2001)

Key Words: prolyl hydroxylase, collagen, enzyme, chondrocytes, endothelial cells


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

PROLYL 4-HYDROXYLASE (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens and more than 15 additional proteins with collagen-like sequences (Kivirikko and Myllyharju 1998 ; Kivirikko and Pihlajaniemi 1998 ). It plays a crucial role in the synthesis of all collagens, because the 4-hydroxyproline residues are essential for the folding of the newly synthesized collagen polypeptide chains into triple-helical molecules (Kivirikko and Myllyharju 1998 ; Kivirikko and Pihlajaniemi 1998 ; Myllyharju and Kivirikko 2001 ).

Vertebrate prolyl 4-hydroxylases are {alpha}2ß2 tetramers in which the ß-subunit is identical to the enzyme and chaperone protein disulfide isomerase (PDI) (Pihlajaniemi et al. 1987 ; Kivirikko and Myllyharju 1998 ; Kivirikko and Pihlajaniemi 1998 ). Recently, an isoform of the {alpha}-subunit, the {alpha}(II)-subunit, was cloned and characterized from mouse (Helaakoski et al. 1995 ) and human (Annunen et al. 1997 ) tissues. This {alpha}-subunit combines with the same PDI polypeptide to form an [{alpha}(II)]2ß2 tetramer, the Type II enzyme (Helaakoski et al. 1995 ; Annunen et al. 1997 ). Correspondingly, the previously known {alpha}-subunit and enzyme tetramer are now called the {alpha}(I) subunit and the Type I enzyme (Helaakoski et al. 1995 ; Annunen et al. 1997 ). The two types of {alpha}-subunit do not appear to co-localize in a single molecule to form a mixed {alpha}(I){alpha}(II)ß2 tetramer (Annunen et al. 1997 ).

Few data are currently available on the expression of the Type I and II prolyl 4-hydroxylase isoenzymes in various cells and tissues. Assays of the respective enzyme activities in cultured cells indicated that the Type I prolyl 4-hydroxylase was the main enzyme form in many cell types but that the Type II enzyme was the main form in chondrocytes (Annunen et al. 1998 ). In agreement with these data, assays in supernatants of homogenates from several mouse tissues indicated that the Type I enzyme was the main form in most tissues studied but that it contributed only slightly more than half of the total prolyl 4-hydroxylase activity in bone and only about 20% of that in cartilage (Annunen et al. 1998 ). Immunofluorescence studies on samples from a fetal human foot confirmed these data and also indicated that the Type II enzyme may represent the main enzyme form in capillary endothelial cells (Annunen et al. 1998 ).

The aim of the present work was to study the expression patterns of the two prolyl 4-hydroxylase isoenzymes in several fetal and adult human tissues, including some malignant tissues. Our data indicate the presence of both spatial and temporal differences in the location of the two isoenzymes, in that Type I is expressed especially by cells of mesenchymal origin and in developing and malignant tissues, whereas Type II is often expressed in more differentiated cells.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Preparation of Antibodies
Immunofluorescence studies (Annunen et al. 1998 ) on the expression patterns of the two types of prolyl 4-hydroxylase isoenzyme were previously performed using monoclonal antibodies L7P to the human {alpha}(I)-subunit and K4 to the mouse {alpha}(II)-subunit (Annunen et al. 1998 ). Two new antibodies were generated for the present work. A polyclonal rabbit antibody to the {alpha}(I)-subunit (R17) was raised against a purified and denatured recombinant human {alpha}(I) polypeptide (Annunen et al. 1997 ), while a monoclonal mouse antibody (M14) to the {alpha}(II)-subunit was generated against a purified and denatured human {alpha}(II) polypeptide (Annunen et al. 1997 ). The polyclonal {alpha}(I) antibody was affinity-purified by diluting the antiserum (1:1) with PBS, pH 7.4, and applying the diluted antiserum to a column containing the Type I prolyl 4-hydroxylase coupled to Sepharose 6B (Pharmacia; Uppsala, Sweden). The column was washed with 2 M NaCl–PBS, pH 7.4, until the A280 decreased to baseline values, and the purified antibodies were then eluted successively by 150 mM glycine-HCl, pH 2.5, and 100 mM trietylamine, pH 11.5, as described by Harlow and Lane 1988 . The fractions containing protein (based on A280) were neutralized, dialyzed against PBS, pH 7.4, and further purified by negative affinity chromatography on a column containing the Type II prolyl 4-hydroxylase coupled to Sepharose 6B (Harlow and Lane 1988 ). The antibodies were finally concentrated using Microsep Microconsentrators (Filter Technology; Northborough, MA). The {alpha}(II) antibody was used either in the form of the nonpurified medium or was purified with Sepharose A. No difference was seen between the results obtained with the nonpurified and purified {alpha}(II) antibodies.

Western Blotting
Cultured cells were harvested, washed twice with PBS, and homogenized in a buffer consisting of 0.1 M NaCl, 0.1 M glycine, 10 µM dithiothreitol, 0.1% Triton X-100, and 0.01 M Tris, pH 7.8, and centrifuged at 10,000 x g for 20 min. Aliquots of the protein extracts were analyzed by nondenaturing PAGE (7.5% gel) in a buffer of 0.192 M glycine, 25 mM Tris, pH 8.3, at 15 V for 32 hr at 4C. For Western analyses, proteins were transferred onto polyvinylidene difluoride membrane (Immobilon P) using the same buffer supplemented with 20% methanol and electrophoresed at 80 V for 1 hr in a cooled chamber. The filters were incubated in a solution of 3% BSA and 0.1% Tween-20 in Tris-buffered saline, pH 7.4 (BSA–TBS), to reduce nonspecific staining. The affinity-purified {alpha}(I) primary antibody was then applied to the filters at a concentration of 1:2000 and incubated for 2 hr at room temperature. The {alpha}(II) antibody was used in the form of the nonpurified monoclonal medium. The filters were washed with TBS, and alkaline phosphate-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibody was applied at a dilution of 1:3000 (Galtac Laboratories; Burlingame, CA). Protein concentrations were determined using the BioRad Protein Assay Kit (Hercules, CA). All experiments were repeated at least three times with essentially identical results.

Immunoelectron Microscopy
The samples were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 hr. Small tissue pieces were immersed in 2.3 M sucrose and frozen in liquid nitrogen, and thin cryosections were cut with a Leica Ultracut UCT microtome. For the single and double immunolabeling, the sections were first incubated in 5% BSA and 0.1% coldwater fish skin (CWFS; Aurion, Wageningen, The Netherlands) gelatin in PBS. The antibodies and gold conjugates were diluted with 0.1% BSA-C (Aurion) in PBS. All washings were performed with 0.1% BSA-C in PBS. For the single-labeling experiment, sections were incubated with the new antibody to the {alpha}(II)-subunit for 60 min. After washing, the sections were exposed to rabbit anti-mouse IgG (Zymed Laboratories; San Francisco, CA) for 30 min, followed by a protein A–gold complex (size 10 nm) for 30 min (Slot and Geuze 1985 ). For the double-labeling experiment, after blocking as described above, sections were exposed to the first primary crossreactive monoclonal {alpha}(II) antibody (Annunen et al. 1998 ) for 60 min, followed by rabbit anti-mouse IgG for 30 min and the protein A–gold complex (size 10 nm) for 30 min. After washing, 1% glutaraldehyde in 0.1 M phosphate buffer was used to block the free binding sites on protein A. The sections were then incubated with the second monoclonal antibody to Type IV collagen (DAKO; Glostrup, Denmark) for 60 min, followed by rabbit anti-mouse IgG for 30 min and a protein A–gold complex (size 5 nm) for 30 min. The controls were prepared by carrying out the labeling procedure without the primary antibody. The sections were embedded in methylcellulose and examined in a Philips CM100 transmission electron microscope.

Immunofluorescence Staining of Tissue Specimens
Specimens from various tissues of an apparently healthy 17-week-old gestational male human fetus (Hagg et al. 1997 ) were available for indirect immunofluorescence studies. In addition, a 10-gestational-week-old placenta obtained from a legal abortion and one full-term placenta were available. Other samples included liver specimens from two healthy adult subjects and three adult patients with hepatocellular carcinoma, which in one case was associated with liver cirrhosis. Samples from two normal adult kidneys, three glomerulonephritis cases, and two kidneys from patients with diabetic glomerulopathy were also studied. Striated muscle and skin samples from an adult subject were also available.

All the tissues had been immediately frozen in liquid nitrogen and stored at -70C. The samples were cut into 5-µm cryosections on SuperFrost glass slides (Mentzel Gläzer; Braunschweig, Germany) and the sections were fixed in precooled methanol for 10 min at -20C. After rinsing with PBS, pH 7.2, nonspecific antibody binding was blocked by incubating the sections with 1% bovine serum albumin (BSA) in PBS, pH 7.2, for 1 hr at 22C, followed by incubation at 4C overnight or 22C for 1 hr with a 1:100 diluted (10 µg/ml) polyclonal antibody to the {alpha}(I)-subunit or 1:25 diluted monoclonal antibody to the {alpha}(II)-subunit. After thorough washing with PBS, a 1:100 diluted tetramethyl rhodamine isothiocyanate (TRITC)-conjugated anti-mouse or anti-rabbit secondary antibody (DAKO) was applied and the sections were incubated in the dark at 4C overnight or at 22C for 1 hr. After washing with PBS, the slides were mounted with glycergel (DAKO) and examined under an epifluorescence microscope (Leitz Aristoplan) equipped with a filter for TRITC fluorescence. Control sections were stained with the secondary antibody alone. For better histological analysis, frozen sections were stained with hematoxylin and eosin by routine methods.

The specificity of stainings was further demonstrated by incubating the affinity-purified polyclonal {alpha}(I) antibody R17 overnight with 2.5 mg of the Type I prolyl 4-hydroxylase and the monoclonal {alpha}(II) antiserum M14 overnight with 2.5 mg of the Type II enzyme, and by using these treated antibodies for immunostaining. No signals were detected in these experiments. The specificity of stainings was also verified by using 1:50 diluted mouse and rabbit non-immunoisotype immunoglobulins (DAKO) as a primary antibody. For additional verification of the results, sections were also stained with a 1:100 diluted antibody to human Type IV collagen (DAKO) and a 1:25 diluted antibody to the endothelial cell marker CD 34 (Novocastra Laboratories; Newcastle, UK). The kidney sections were also stained with a 1:80 diluted proximal tubule cell marker gb200 (NeoMarkers; Fremont, CA) and the skin samples with a 1:100 diluted monoclonal antibody to the human PDI polypeptide (DAKO), a 1:50 diluted {alpha}-smooth muscle antibody (NeoMarkers) and a 1:50 diluted anti-keratin 7 antibody.

Cultured Cells and Their Immunofluorescence Staining
Human umbilical vein endothelial cells (Edgell et al. 1983 ) and human hepatoblastoma cells (line HepG2, a kind gift from Dr. Orlando Musso) were cultured on glass plates. The endothelial cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Lexington, KY) containing 10% fetal bovine serum, 2 mM glutamine, 100 mM sodium pyruvate, and 1 x HAT as described by Edgell et al. 1983 . The HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum (Sigma; St Louis, MO) under standard conditions.

Mouse chondrocytes were obtained from the heads of the ribs of 7-day-old mice and human chondrocytes from a surgical limb amputation. The cells were fixed in precooled methanol at -20C for 10 min and washed three times with PBS. Nonspecific antibody binding was blocked by incubating the plates in 2% BSA–PBS for 1 hr. The samples were then incubated for 2 hr at room temperature with a 1:100 (10 µg/ml) diluted antibody to the {alpha}(I)-subunit or an undiluted monoclonal antibody pool for the {alpha}(II)-subunit. The samples were washed again with PBS and incubated with a 1:100 diluted TRITC-conjugated anti-mouse or anti-rabbit secondary antibody. Control sections were stained with a 1:100 diluted monoclonal antibody to the human PDI polypeptide and a 1:50 diluted anti-keratin 7 antibody, the antigen-blocked antibodies to the {alpha}(I)- and {alpha}(II)-subunits as described above, and the mouse and rabbit non-isotype immunoglobulins described above.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Generation of New Antibodies
The MAb K4 to the {alpha}(II)-subunit used previously had been found to give a weak signal in the basement membrane zone below the epidermis in the samples from a fetal foot (Annunen et al. 1998 ). Our initial experiments showed staining in several additional basement membrane zones (details not shown). When immunoelectron microscopy was used to verify the specificity of this staining, the antibody was found to give a signal in the endoplasmic reticulum of the capillary endothelial cells (Fig 1A) consistent with the known location of the prolyl 4-hydroxylase tetramer in the endoplasmic reticulum (Kivirikko and Myllyharju 1998 ; Kivirikko and Pihlajaniemi 1998 ). However, the antibody also recognized a basement membrane component outside the cells (Fig 1A), even though prolyl 4-hydroxylase is believed to be only an intracellular enzyme (Kivirikko and Myllyharju 1998 ; Kivirikko and Pihlajaniemi 1998 ). The basement membrane signal appeared quite specific and consisted of clusters of label in close contact with the endothelial cell membrane.



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Figure 1. Immunoelectron microscopy of a capillary endothelial cell in a muscle tissue. The previously used monoclonal antibody K4 to the {alpha}(II)-subunit (Annunen et al. 1998 ) (A) gave a signal in the endoplasmic reticulum of capillary endothelial cells in double-labeling experiments and also in the basement membrane outside the cells (arrowheads). An antibody to Type IV collagen stained only the basement membrane (arrow). The new MAb M14 to the {alpha}(II)-subunit (B) stained the endoplasmic reticulum of a capillary endothelial cell (arrowhead), whereas the basement membrane remained unlabeled. Gold particle size for Type IV collagen antibody is 5 nm and for {alpha}(II) antibody 10 nm. Bars = 0.2 µm.

To study this signal further, a new MAb M14 to the human {alpha}(II)-subunit was generated for the present work. A polyclonal antibody (R17) to the {alpha}(I)-subunit was also generated and was found to give a slightly stronger signal than the previously used MAb (Annunen et al. 1998 ). The new MAb M14 was found to recognize both the native Type II prolyl 4-hydroxylase and the denatured {alpha}(II)-subunit in EIA (details not shown) but only the native enzyme in Western blotting (Fig 2A, Lane 2). No staining of the Type I enzyme (Fig 2A, Lane 1) or the {alpha}(I)-subunit was seen in any control experiments (details not shown). In the immunofluorescence experiments, the antibody recognized both the human and mouse Type II enzyme. The new polyclonal antibody R17 to the {alpha}(I)-subunit recognized both the native Type I enzyme (Fig 2B, Lane 1) and the denatured {alpha}(I)-subunit in Western blotting, and showed no crossreactivity with the Type II enzyme (Fig 2B, Lane 2) or the denatured {alpha}(II)-subunit (details not shown).



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Figure 2. Characterization of the new antibodies to prolyl 4-hydroxylase isoenzymes by Western blotting of samples separated by PAGE under nondenaturing conditions. The Western blotting was performed with either the MAb M14 to the {alpha}(II)-subunit (A) or the polyclonal antibody R17 to the {alpha}(I)-subunit (B). The samples analyzed were purified human Type I prolyl 4-hydroxylase tetramer [{alpha}(I)]2ß2 (Lanes 1), purified human Type II prolyl 4-hydroxylase tetramer [{alpha}(II)]2ß2 (Lanes 2), and soluble proteins from cultured human umbilical vein endothelial cells (Lanes 3), mouse chondrocytes (Lanes 4), and human chondrocytes (Lanes 5).

The Type II Prolyl 4-hydroxylase Is the Main or Only Enzyme Form in Capillary Endothelial Cells and Cultured Umbilical Vein Endothelial Cells
The new MAb M14 to the {alpha}(II)-subunit, like the previously used antibody (see above), gave a signal in the endoplasmic reticulum of capillary endothelial cells in immunoelectron microscopy but no signal in the basement membrane outside the cells (Fig 1B).

The specificity of the endothelial cell signal was studied further by immunofluorescence staining of cultured human umbilical vein endothelial cells. A strong signal was obtained both with the previously used (Fig 3A) K4 antibody to the {alpha}(II)-subunit (Annunen et al. 1998 ) and the new M14 antibody (Fig 3B), whereas no staining was seen with the antibody to the {alpha}(I)-subunit (data not shown). The specificity of this staining was studied further by Western blotting analyses of non-denatured proteins from the cultured endothelial cells. Both the previously used K4 antibody (Annunen et al. 1998 ) (data not shown) and the new M14 antibody recognized a protein with a mobility identical to that of the prolyl 4-hydroxylase tetramer (Fig 2A, Lane 3), whereas no staining was detected with the R17 antibody to the {alpha}(I)-subunit (Fig 2B, Lane 3). The antibodies to the {alpha}(II)- and {alpha}(I)-subunits both recognized the enzyme tetramer in Western blots of cultured mouse (Fig 2A and Fig 2B, Lane 4) and human (Fig 2A and Fig 2B, Lane 5) chondrocytes. The slightly slower mobility of the Type I enzyme in chondrocytes may be due to a more extensive glycosylation of the {alpha}(I)-subunit in these cells.



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Figure 3. Staining of cultured human umbilical vein endothelial cells with the K4 (A) and M14 (B) antibodies to the {alpha}(II)-subunit. Bars: A = 50 µm; B = 20 µm.

Type II Prolyl 4-hydroxylase Has an Important Role in Kidney Development
In metanephric blastema of the fetal kidney, there were occasional primitive tubular structures showing positivity for the Type II subunit (Fig 4A) and the cells of the developing glomeruli were also faintly positive (Fig 4C and Fig 4D). Endothelial cells of blood vessels, regardless of the size of the vessel, stained positively, and the staining was essentially similar in the fetal and adult kidney. Tubular structures of collecting duct caliber stained positively for the {alpha}(II)-subunit in both fetal and mature kidneys (Fig 4B), although the intensity of staining was weaker in the mature kidney.



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Figure 4. Expression of prolyl 4-hydroxylase isoenzymes in the developing and adult kidney. Tubular structures of collecting duct caliber in both the fetal (A) and adult (B) kidney stained positively with the {alpha}(II) antibody M14 (arrows). Developing glomeruli at various maturation stages (C,D) were also positive in staining with the M14 antibody (arrows). Undifferentiated mesenchymal cells of the developing interstitium in the fetal kidney (E) became stained with the antibody R17 to the {alpha}(I)-subunit (arrow). In adult kidney (F), the glomerular mesangial cells (arrowhead) and smooth muscle cells of the arterial walls (arrow) gave a signal with the R17 antibody. Staining of adult kidney sections with antigen-blocked antibodies (see Materials and Methods) to the {alpha}(I)- (G) and {alpha}(II)- (H) subunits is also shown. Bars: F = 20 µm; A–E,G,H 50 µm.

The expression of the Type I enzyme differed from that of the Type II enzyme. In the fetal kidney, a clear signal for the Type I enzyme was present in the undifferentiated mesenchymal cells of the developing interstitium (Fig 4E), some immature tubules, and the capsule, and a faint signal was also seen among metanephric blastemata in the developing kidney. In the adult kidney, the Type I enzyme was expressed in interstitial fibroblasts around tubular structures, in fibroblastic cells of the capsule and in smooth muscle cells of arterial walls, and faintly in mesangial cells of the glomeruli (Fig 4F). The expression patterns of the two types of prolyl 4-hydroxylase isoenzyme in samples from the kidneys with diabetic nephropathy and glomerulonephritis were essentially identical to those seen in the healthy adult kidney (details not shown).

Expression Patterns of the Prolyl 4-hydroxylase Isoenzymes in Fetal and Adult Liver
Staining of the fetal liver samples with the {alpha}(II) antibody was generally faint, the strongest signal being seen in the periportal hepatocytes of the developing liver (Fig 5A). In the adult liver, occasional sinusoidal structures became stained with the {alpha}(II) antibody (Fig 5B), and a positive signal was also seen in the epithelium of the bile ducts. As in other tissues, the capillaries became stained with the {alpha}(II) antibody.



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Figure 5. Expression of prolyl 4-hydroxylase isoenzymes in the liver. In the fetal liver (A), the periportal hepatocytes (arrow) became stained with the M14 antibody to the {alpha}(II)-subunit. In the adult liver (B), some sinusoidal structures, the epithelium of the bile ducts, and the endothelium of the portal capillaries (arrow) were stained with the M14 antibody (P, portal area). The antibody R17 to the {alpha}(I)-subunit stained some hepatocytes, and there was also faint staining in sinusoidal structures (arrow) in the adult liver (C). Malignant hepatocytes in hepatocellular carcinoma became stained with the antibodies to both the {alpha}(II)-subunit (M14) (D) and the {alpha}(I)-subunit (R17) (E), the signal for the Type I enzyme being far stronger. Cultured hepatoblastoma cells gave a strong signal with the R17 antibody (F) and a faint signal with the M14 antibody (G). Bars = 50 µm.

The antibody to the {alpha}(I)-subunit stained fibroblastic cells and smooth muscle cells in the portal tracts of the fetal liver but gave no detectable signal in the parenchyma. Staining with this antibody was seen, however, in some hepatocytes in the adult liver (Fig 5C). The {alpha}(I) antibody also stained the capsular fibroblasts.

Examination of liver specimens from patients with hepatocellular carcinoma with or without cirrhosis showed the malignant hepatocytes to give a signal for the Type II enzyme (Fig 5D), although the signal for Type I was far stronger (Fig 5E). A strong signal was seen in the fibroblasts of the cirrhotic area and in the smooth muscle cells of larger vessels with the {alpha}(I) antibody. Cultured hepatoblastoma cells became strongly stained with the antibodies to the {alpha}(I)-subunit (Fig 5F) and the PDI polypeptide, i.e., the ß-subunit: (data not shown), whereas only a very faint signal was obtained with the antibody to the {alpha}(II)-subunit (Fig 5G). An {alpha}-smooth muscle antibody and a secondary antibody alone (details not shown) showed only background staining.

Skeletal Myocytes Are Stained Only with the {alpha}(I) Antibody
The antibody to the {alpha}(II)-subunit stained the capillaries of adult human striated muscle, whereas the myocytes remained negative (Fig 6A). These were strongly stained with the {alpha}(I) antibody (Fig 6B), however, and the smooth muscle cells of the large arteries also gave a signal with this antibody. In the skin, the arrector muscle of the hair gave a strong signal with the {alpha}(I) antibody (Fig 6C).



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Figure 6. Expression of prolyl 4-hydroxylase isoenzymes in muscle. The capillaries in an adult striated muscle became stained with the {alpha}(II) antibody M14 (arrow), whereas the myocytes (M) were negative (A) and were stained with the {alpha}(I) antibody R17 (B). The arrector muscle of a hair (arrow) gave a strong signal with R17 for the {alpha}(I)-subunit and the fibroblasts (F) around this muscle likewise became stained (C). Bars = 50 µm.

Prolyl 4-hydroxylase Isoenzymes Show Different Expression Patterns in Placenta
The villous capillaries of the early placenta (gestational Week 9) were clearly stained with the {alpha}(II) antibody (Fig 7A), but otherwise the villous stromal myofibroblasts were negative, as was the villous trophoblastic epithelium. The decidual cells were either negative or became only faintly stained with the {alpha}(II) antibody, but occasional cells in the decidual membrane became strongly stained (Fig 7A). These probably represent invasive cytotrophoblasts, i.e., intermediate trophoblasts. The {alpha}(II) staining pattern in the full-term placenta was similar to that in the early placenta except that the staining was somewhat weaker.



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Figure 7. Expression of prolyl 4-hydroxylase isoenzymes in placenta. The villous capillaries (arrow) of the early placenta became stained with the {alpha}(II) antibody M14, whereas the villous stromal myofibroblasts and villous trophoblastic epithelium were negative (A). The antibody R17 to the {alpha}(I)-subunit stained the stromal fibroblasts of the stem villi strongly (B), while the villous stromal fibroblasts of term villi were either weakly positive or negative. The decidual cells became clearly stained with the R17 antibody, but the smooth muscle cells of the spiral arteries were relatively weakly stained (C). Occasional cells in the cytotrophoblastic columns of the developing placenta became strongly stained for both M14 (D) and R17 (E) antibodies. Staining with the antibody-blocked R17 antibody (see Materials and Methods) is also shown (H). Bars = 50 µm.

The staining pattern obtained with the {alpha}(I) antibody was different from that with the {alpha}(II) antibody, the strongest staining being seen in the stromal myofibroblasts of the stem villi (Fig 7B). Otherwise, the villous stromal fibroblasts were either weakly positive or negative. In the decidua, the decidual cells and invasive cytotrophoblasts became clearly stained with the {alpha}(I) antibody (Fig 7C). The smooth muscle cells of the spiral arteries showed relatively weak staining, whereas the periarterial fibroblastic cells were strongly stained (Fig 7C). The endothelial cells of the capillaries and endometrial glands were negative. Occasional cells in the cytotrophoblastic columns became strongly stained with both the {alpha}(II) and {alpha}(I) antibodies (Fig 7D and Fig 7E).

Prolyl 4-hydroxylase Expression in Developing Bone Appears to Be Developmentally Regulated
When studying enchondral ossification previously, we found that the Type I prolyl 4-hydroxylase is expressed in the ossification process earlier than the Type II enzyme (Annunen et al. 1998 ). We now studied whether this was also the case in intramembranous ossification in a 17-week-old fetal calvaria. We regarded it possible that, because in intramembranous ossification the bone forms via the condensation and differentiation of mesenchymal cells in the osteoid secreted by osteoblasts, the developing bone might stain almost exclusively with the {alpha}(I) antibody. However, we found that the osteoblasts stained strongly with both the {alpha}(I) antibody (Fig 8A) and the {alpha}(II) antibody (Fig 8B). The signal for the {alpha}(I) antibody was seen earlier in ossification, i.e., even in the undifferentiated mesenchymal cells, whereas the signal for the {alpha}(II) antibody became evident only later during ossification. The capillaries stained with the antibody to the {alpha}(II)-subunit, as in the other tissues.



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Figure 8. Expression of prolyl 4-hydroxylase isoenzymes in the developing calvaria. The antibody R17 to the {alpha}(I)-subunit (A) and M14 to the {alpha}(II)-subunit (B) both gave a strong signal in the fetal osteoblasts (arrows). Bars: A = 50 µm; B = 20 µm.

Control staining experiments on a 17-week-old fetal humerus indicated that the new M14 antibody to the {alpha}(II)-subunit gave a staining pattern identical to that previously reported (Annunen et al. 1998 ) for the K4 antibody in enchondral ossification (details not shown).


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Prolyl 4-hydroxylase had long been assumed to be of one type only, with no isoenzymes, until the {alpha}(II)-subunit was cloned from mouse (Helaakoski et al. 1995 ) and human (Annunen et al. 1997 ) and the corresponding enzyme tetramers were characterized (Helaakoski et al. 1995 ; Annunen et al. 1997 ). The Type I and Type II isoenzymes have highly similar catalytic properties but there are distinct, although small, differences in their Km values for various peptide substrates and major differences in their Ki values for the competitive inhibitor poly(L-proline) (Helaakoski et al. 1995 ; Annunen et al. 1997 ; Myllyharju and Kivirikko 1999 ).

The only previous report (Annunen et al. 1998 ) and the present data (as summarized in Table 1) indicate that the two prolyl 4-hydroxylase isoenzymes have major differences in their expression patterns in many tissues. The Type I enzyme was previously found to be the main form in many cells and tissues, but the Type II enzyme was the main form in cultured chondrocytes and chondrocytes present in a fetal cartilage (Annunen et al. 1998 ).


 
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Table 1. Immunofluorescence staining of various cell types with antibodies to the {alpha}(I)- and {alpha}(II)-subunits of prolyl 4-hydroxylasea

Immunofluorescence staining experiments further suggested that the Type II enzyme may be the main form in capillary endothelial cells (Annunen et al. 1998 ). The present data indicate that the signal found around the capillaries in our previous study (Annunen et al. 1998 ) was due in part to staining of an extracellular basement membrane component. No explanation is currently available for this basement membrane signal. It is well established that prolyl 4-hydroxylase is found only in the endoplasmic reticulum of various cells (Kivirikko and Myllyharju 1998 ; Kivirikko and Pihlajaniemi 1998 ), and therefore the extracellular signal must be due to some basement membrane component that crossreacted with MAb K4 but not with MAb M14.

Nevertheless, our current work verifies the expression of the Type II enzyme in capillary endothelial cells and extends the previous findings by demonstrating this localization in the capillaries of many tissues and also in cultured umbilical vein endothelial cells. Furthermore, our present data indicate that these endothelial cells appear to differ from chondrocytes in that they express very little, if any, Type I prolyl 4-hydroxylase, because no signal for this isoenzyme was seen in any tissue even with the new {alpha}(I) antibody, and no staining was detected even in immunostaining or Western blotting of the cultured umbilical vein endothelial cells.

The Type II prolyl 4-hydroxylase was also the main isoenzyme in many other cell types, such as cells of developing glomeruli in their vesicular and later developmental stages and in tubular structures of collecting duct caliber both in the fetal and adult kidney. In the liver, the Type II enzyme was additionally found in occasional sinusoidal structures and in the epithelium of the bile ducts, and in the placenta it was found not only in the capillary endothelial cells but also in some cells of the decidual membrane that probably represented invasive cytotrophoblasts. Interestingly, osteoblasts stained strongly for the Type II isoenzyme in addition to their strong staining for the Type I isoenzyme, even in the case of a fetal calvaria, e.g., in a bone developing by intramembranous ossification.

The Type I isoenzyme appeared to be the main form or only form in undifferentiated fibroblastoid mesenchymal cells of the developing kidney interstitium and bone, e.g., in fibroblasts and fibroblastic cells in many tissues and in large decidual cells and infiltrating trophoblastic cells in the placenta. Additional cell types that expressed the Type I isoenzyme include skeletal myocytes and smooth muscle cells, which may have this isoenzyme as their only prolyl 4-hydroxylase form, as well as osteoblasts, which express both isoenzymes strongly, and chondrocytes (Annunen et al. 1998 ), which have the Type II enzyme as their main form.

Considered collectively, the data suggest that Type I prolyl 4-hydroxylase is expressed especially by cells of mesenchymal origin, and it may often be present in less differentiated cells than the Type II isoenzyme. Hepatocytes represent an exception to this rule, in that they do express small amounts of the Type I isoenzyme and express little if any Type II isoenzyme. The Type II isoenzyme is likewise expressed, at least in small amounts, by many mesenchyme-derived cells (our previous and present data), but especially by chondrocytes and endothelial cells (Annunen et al. 1998 ). Epithelial cells appeared to express only small amounts of either prolyl 4-hydroxylase form in the tissues studied here, the main or only isoenzyme in the epithelial cells of the collecting ducts in the kidney and the bile ducts being Type II. The parenchymal cells in the adult liver expressed low but detectable levels of the Type I isoenzyme, but this signal was much stronger in malignant hepatocytes and very strong in cultured hepatoblastoma cells. These findings agree with the suggestion that the Type I isoenzyme may be highly expressed in less differentiated cells, including malignant hepatocytes. The increased expression level found in malignant hepatocytes may well be related to an increased rate of collagen synthesis by these cells, because a large number of previous studies have demonstrated that the levels of total prolyl 4-hydroxylase activity in various cell types usually increase and decrease with changes in the rate of collagen synthesis (Kivirikko and Pihlajaniemi 1998 ).

The collagen family consists of more than 20 proteins formally defined as collagens and more than 15 additional proteins with collagen-like domains (Kielty et al. 1993 ; Myllyharju and Kivirikko 2001 ). All these proteins contain a number of 4-hydroxyproline residues that have been formed from proline residues in peptide linkages by the action of prolyl 4-hydroxylases (Kivirikko and Myllyharju 1998 ; Myllyharju and Kivirikko 2001 ). The existence of two forms of prolyl 4-hydroxylase raises the possibility that these isoenzymes may show different specificities with respect to the collagen types to be hydroxylated, but no direct data are available to elucidate this possibility. If the Type II isoenzyme had been expressed only in chondrocytes, it might well have been specific for cartilage collagens, i.e., Types II, IX, and XI, and possibly type X, which is synthesized by hypertrophic chondrocytes (Kielty et al. 1993 ; Myllyharju and Kivirikko 2001 ). The present data indicating that this isoenzyme is the main form in many other cell types as well nevertheless argue against such a possibility. Both isoenzymes hydroxylate proline residues in -X-Pro-Gly- triplets, in which X can be almost any amino acid (Kivirikko and Myllyharju 1998 ; Kivirikko and Pihlajaniemi 1998 ). Because all collagens and related proteins contain similar triplets, although not in identical sequences, it is obvious that both isoenzymes must be able to hydroxylate identical triplets. It therefore appears that the main differences between the isoenzymes may be found in their expression patterns in various cells rather than their specificities with respect to various collagen types. Nevertheless, the isoenzymes may well also show at least minor differences in their specificities with respect to the collagen types to be hydroxylated, and studies should be performed to elucidate this possibility. It should also be noted that some cell types that are likely to synthesize collagens expressed only very small amounts of either prolyl 4-hydroxylase isoenzyme. This finding raises the possibility that there may exist a further isoenzyme(s) to be identified in the future.


  Acknowledgments

Supported by grants from the Health Sciences Council of the Academy of Finland, from the Finnish Centre of Excellence Programme 2000-2005 (44843), and from FibroGen Inc. (South San Francisco, CA).

We thank Liisa Äijälä and Annikki Huhtela for their expert technical assistance and Timo Väisänen for helping with problems concerning the microscopy and the histological samples.

Received for publication November 30, 2000; accepted March 28, 2001.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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