ARTICLE |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 49:11431153, 2001)
Key Words: prolyl hydroxylase, collagen, enzyme, chondrocytes, endothelial cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (
Vertebrate prolyl 4-hydroxylases are 2ß2 tetramers in which the ß-subunit is identical to the enzyme and chaperone protein disulfide isomerase (PDI) (
-subunit, the
(II)-subunit, was cloned and characterized from mouse (
-subunit combines with the same PDI polypeptide to form an [
(II)]2ß2 tetramer, the Type II enzyme (
-subunit and enzyme tetramer are now called the
(I) subunit and the Type I enzyme (
-subunit do not appear to co-localize in a single molecule to form a mixed
(I)
(II)ß2 tetramer (
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 (
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of Antibodies
Immunofluorescence studies ((I)-subunit and K4 to the mouse
(II)-subunit (
(I)-subunit (R17) was raised against a purified and denatured recombinant human
(I) polypeptide (
(II)-subunit was generated against a purified and denatured human
(II) polypeptide (
(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 NaClPBS, 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
(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
(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 (BSATBS), to reduce nonspecific staining. The affinity-purified (I) primary antibody was then applied to the filters at a concentration of 1:2000 and incubated for 2 hr at room temperature. The
(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 (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 Agold complex (size 10 nm) for 30 min (
(II) antibody (
Immunofluorescence Staining of Tissue Specimens
Specimens from various tissues of an apparently healthy 17-week-old gestational male human fetus (
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 (I)-subunit or 1:25 diluted monoclonal antibody to the
(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 (I) antibody R17 overnight with 2.5 mg of the Type I prolyl 4-hydroxylase and the monoclonal
(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
-smooth muscle antibody (NeoMarkers) and a 1:50 diluted anti-keratin 7 antibody.
Cultured Cells and Their Immunofluorescence Staining
Human umbilical vein endothelial cells (
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% BSAPBS 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 (I)-subunit or an undiluted monoclonal antibody pool for the
(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
(I)- and
(II)-subunits as described above, and the mouse and rabbit non-isotype immunoglobulins described above.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of New Antibodies
The MAb K4 to the (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 (
|
To study this signal further, a new MAb M14 to the human (II)-subunit was generated for the present work. A polyclonal antibody (R17) to the
(I)-subunit was also generated and was found to give a slightly stronger signal than the previously used MAb (
(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
(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
(I)-subunit recognized both the native Type I enzyme (Fig 2B, Lane 1) and the denatured
(I)-subunit in Western blotting, and showed no crossreactivity with the Type II enzyme (Fig 2B, Lane 2) or the denatured
(II)-subunit (details not shown).
|
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 (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 (II)-subunit (
(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 (
(I)-subunit (Fig 2B, Lane 3). The antibodies to the
(II)- and
(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
(I)-subunit in these cells.
|
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 (II)-subunit in both fetal and mature kidneys (Fig 4B), although the intensity of staining was weaker in the mature kidney.
|
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 (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
(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
(II) antibody.
|
The antibody to the (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
(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 (I) antibody. Cultured hepatoblastoma cells became strongly stained with the antibodies to the
(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
(II)-subunit (Fig 5G). An
-smooth muscle antibody and a secondary antibody alone (details not shown) showed only background staining.
Skeletal Myocytes Are Stained Only with the (I) Antibody
The antibody to the (II)-subunit stained the capillaries of adult human striated muscle, whereas the myocytes remained negative (Fig 6A). These were strongly stained with the
(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
(I) antibody (Fig 6C).
|
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 (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
(II) antibody, but occasional cells in the decidual membrane became strongly stained (Fig 7A). These probably represent invasive cytotrophoblasts, i.e., intermediate trophoblasts. The
(II) staining pattern in the full-term placenta was similar to that in the early placenta except that the staining was somewhat weaker.
|
The staining pattern obtained with the (I) antibody was different from that with the
(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
(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
(II) and
(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 ((I) antibody. However, we found that the osteoblasts stained strongly with both the
(I) antibody (Fig 8A) and the
(II) antibody (Fig 8B). The signal for the
(I) antibody was seen earlier in ossification, i.e., even in the undifferentiated mesenchymal cells, whereas the signal for the
(II) antibody became evident only later during ossification. The capillaries stained with the antibody to the
(II)-subunit, as in the other tissues.
|
Control staining experiments on a 17-week-old fetal humerus indicated that the new M14 antibody to the (II)-subunit gave a staining pattern identical to that previously reported (
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Prolyl 4-hydroxylase had long been assumed to be of one type only, with no isoenzymes, until the (II)-subunit was cloned from mouse (
The only previous report (
|
Immunofluorescence staining experiments further suggested that the Type II enzyme may be the main form in capillary endothelial cells (
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 (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 (
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 (
The collagen family consists of more than 20 proteins formally defined as collagens and more than 15 additional proteins with collagen-like domains (
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Annunen P, AutioHarmainen H, Kivirikko KI (1998) The novel type II prolyl 4-hydroxylase is the main enzyme form in chondrocytes and capillary endothelial cells, whereas the type I enzyme predominates in most cells. J Biol Chem 273:5989-5992
Annunen P, Helaakoski T, Myllyharju J, Veijola J, Pihlajaniemi T, Kivirikko KI (1997) Cloning of the human prolyl 4-hydroxylase subunit isoform
(II) and characterization of the type II enzyme tetramer. The
(I) and
(II) subunits do not form a mixed
(I)
(II)ß2 tetramer. J Biol Chem 272:17342-17348
Edgell CJS, McDonald CC, Graham JB (1983) Permanent cell line expressing factor VIII related antigen established by hybridization. Proc Natl Acad Sci USA 80:3734-3737[Abstract]
Hägg PM, Hägg PO, Peltonen S, AutioHarmainen H, Pihlajaniemi T (1997) Location of type XV collagen in human tissues and its accumulation in the interstitial matrix of fibrotic kidney. Am J Pathol 150:2075-2086[Abstract]
Harlow E, Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press
Helaakoski T, Annunen P, Vuori K, MacNeil IA, Pihlajaniemi T, Kivirikko KI (1995) Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase -subunit isoform: formation of an
2ß2 tetramer with the protein disulfide isomerase/ß subunit. Proc Natl Acad Sci USA 92:4427-4431[Abstract]
Kielty CM, Hopkinson I, Grant ME (1993) Collagen: the collagen family: structure, assembly and organization in the extracellular matrix. In Royce PM, Steinmann B, eds. Connective Tissue and Its Heritable Disorders. Molecular, Genetics, and Medical Aspects. New York, WileyLiss, 103-147
Kivirikko KI, Myllyharju J (1998) Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. Matrix Biol 16:357-368[Medline]
Kivirikko KI, Pihlajaniemi T (1998) Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases. Adv Enzymol Relat Areas Mol Biol 72:325-398[Medline]
Myllyharju J, Kivirikko KI (1999) Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase. EMBO J 18:306-312
Myllyharju J, Kivirikko KI (2001) Collagens and collagen-related diseases. Ann Med 33:7-21[Medline]
Pihlajaniemi T, Helaakoski T, Tasanen K, Myllylä R, Huhtala ML, Koivu J, Kivirikko KI (1987) Molecular cloning of the ß-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are the products of the same gene. EMBO J 6:643-649[Abstract]
Slot JW, Geuze HJ (1985) For double labeling experiments. A novel method to make gold probes for multiple labeling cytochemistry. Eur J Cell Biol 38:87-93[Medline]