CELL STRUCTURE AND FUNCTION 34: 97–104 (2009) © 2009 by Japan Society for Cell Biology

Expression of Prolyl 3-hydroxylase Genes in Embryonic and Adult Mouse Tissues

Janice Vranka1, H. Scott Stadler1,2, and Hans Peter Bächinger1,3∗ 1Research Department, Shriners Hospital for Children, Portland, Oregon, 2Oregon Health and Science University, Department of Molecular and Medical Genetics, Portland, Oregon, 3Oregon Health and Science University, Department of Biochemistry and Molecular Biology, Portland, Oregon

ABSTRACT. requires hydroxylation of its proline residues to achieve proper assembly, structure, and function. Prolyl 4-hydroxylase catalyzes formation of 4-hydroxyproline, which is essential for collagen triple helix formation and stability. Prolyl 3-hydroxylase catalyzes formation of 3-hydroxyproline, which is far less abundant in and whose function remains unclear. Recently mutations in prolyl 3-hydroxylase 1 have been associated with , yet the temporal and spatial expression patterns of the prolyl 3- hydroxylase family members during development and in adult tissues remain undefined. By northern blot analysis distinct differences in transcript sizes of the three prolyl 3-hydroxylase genes were detected. Quantitative RTPCR demonstrated tissue-specific differences in prolyl 3-hydroxylase expression, most notable of which were high levels of prolyl 3-hydroxylase 2 in kidney and prolyl 3-hydroxylase 1 expression in embryonic tissues. Finally, in situ hybridization was used to assess spatio-temporal distribution of three prolyl 3-hydroxylases at embryonic days 11–15. Importantly, prolyl 3-hydroxylase 1 was expressed within condensations of the vertebral bodies and in the aortic arch of the developing heart, whereas prolyl 3-hydroxylase 2 was expressed in developing lens capsule. The prolyl 3-hydroxylase 3 gene showed more generalized expression overlapping somewhat with the other two genes. This report characterizes expression of the three prolyl 3-hydroxylase genes in embryonic and adult mice. Overall these data demonstrate tissue specific prolyl 3-hydroxylase gene expression in both fetal and adult tissues indicating a developmental role for prolyl 3-hydroxylase activity.

Key words: prolyl hydroxylase/3-hydroxyproline/collagen/posttranslational modifications

Introduction tion varies with the different types of collagens and occurs in the largest amounts in collagen types IV and V Prolyl hydroxylation is known to be a critical post transla- (Kefalides, 1972, 1973; Rhodes and Miller, 1978). Impor- tional event during collagen biosynthesis and is catalyzed tantly, prolyl 3-hydroxylase 2 was recently shown to prefer- by multiple members of the iron-dependent dioxygenase entially hydroxylate two synthetic peptides corresponding superfamily. Collagen prolyl 4-hydroxylase catalyzes the to sequences that are hydroxylated in type IV collagen formation of 4-hydroxyproline in -X-Pro-Gly- triplets in (Tiainen et al., 2008). collagens and prolyl 3-hydroxylase 1 (P3H1) has been The prolyl 3 hydroxylase family of consists of 3 shown to catalyze the formation of 3-hydroxyproline in members, all of which share the conserved residues of the procollagens (Vranka et al., 2004). Interestingly, although active site domain of the iron-dependent dioxygenases, 4(R)-hydroxyproline is clearly required for proper assembly including those of the prolyl 4-hydroxylases and lysyl and folding of the collagen triple helix, the importance of hydroxylases. Additionally, P3H1 has been shown to spe- 3(S)-hydroxyproline is only just beginning to be elucidated. cifically interact with cartilage associated (CRTAP) 3-hydroxyproline is found in almost all collagens in the and cyclophilin B (Vranka et al., 2004). CRTAP is a protein sequence -Gly-3Hyp-4Hyp-Gly- (Fietzek et al., 1973, 1972; of unknown function that shares homology with the amino Rexrodt et al., 1975); however, the extent of 3-hydroxyla- terminal domain of the P3H family of proteins (Morello et al., 1999; Tonachini et al., 1999; Vranka et al., 2004). *To whom correspondence should be addressed: Hans Peter Bächinger, Multiple recent reports have shown that P3H1 deficiency in Research Department, Shriners Hospital for Children, 3101 SW Sam Jackson humans, as well as mutations in CRTAP, causes a recessive Park Road, Portland, OR 97239. E-mail: [email protected] disorder resembling osteogenesis imperfecta (OI) (Barnes et Grant sponsor: Shriners Hospital for Children Research (HPB & HSS) al., 2006; Cabral et al., 2007; Morello et al., 2006; Willaert

97 J. Vranka et al. et al., 2008). More importantly, the 3-hydroxylation of key turer’s instructions with Oligo(dT)12–18 primer (Invitrogen). Addi- residues in types I and II collagens was reduced in the tional RNA was extracted from adult mouse bones, joint patients with OI indicating the importance of P3H1 and and skin using the TRIzol extraction method and reverse tran- CRTAP in collagen stability, secretion, and ultimately in scribed using random hexamers according to the manufacturer’s bone development. instructions (Invitrogen). Resulting cDNA was used as the tem- The aim of the present work was to study the expression plate in real-time quantitative PCR experiments with the same P3H patterns of the three prolyl 3-hydroxylase genes in adult and 1, 2, or 3 gene specific oligonucleotides described above, and fetal mouse tissues in order to determine in vivo sites the iQ SYBR Green Supermix according to the manufacturer’s expressing the prolyl 3-hydroxylase enzymes and to iden- instructions (Bio-Rad). Quantitative PCR data were measured and tify tissues most likely to be affected by their loss of func- analyzed using the iQ5 Multicolor Real-Time PCR Detection tion. System and software version 2.0 (Bio-Rad). The following gene specific oligonucleotides to 18srRNA were synthesized 5'CAGT- AAGTGCGGGTCATAAGC and 5'AGGGCCTCACTAAACCA- Materials and Methods TCC and amplified a 96 bp product, and additional PCR experi- ments were performed using the 18srRNA primers and the same Northern blots of mouse tissues GAPDH sequences as mentioned above as reference genes for data normalization. Each PCR reaction was 25 μl and apart from that Mouse multiple tissue Northern blots containing 2 μg of poly A+ used for the standard curves, the total input cDNA was identical RNA per lane were purchased from BD Biosciences (Clontech). for all reactions. Because SYBR Green binding is not sequence Hybridization was carried out at 60°C using express hybridization specific, careful validation of the primer pairs was undertaken to buffer (BD Biosciences). The membranes were washed 4×20 min ensure that only the target gene sequence was amplified. To verify in 2x sodium chloride-sodium citrate buffer (SSC) and 1% SDS at the specificity of each amplicon, a single gel band of the expected room temperature, then washed in 1x SSC and 0.1% SDS twice size was amplified for each primer pair when tested on a mouse and autoradiographed. 3'UTR probes of approximately 600 nucle- fibroblast cDNA and melting curve analysis confirmed the pres- otides in length were PCR-amplified from mouse fibroblast cDNA ence of a single PCR product. Standard curves were generated for for P3H1 and P3H2 probes with the following gene specific oligo- each amplified product and data obtained in all experiments were nucleotides: for the P3H1 probe 5'AGGCAGATGACCTGGT- confirmed to correspond to a linear portion of the standard curve GAAG and 5'GCCTACATAGGCCCCAGGGTGG; for the P3H2 generated for each experiment. Reaction efficiencies determined probe 5'ATTCAGAGCCCATTCACACC and 5'TTAATCCAGT- from calibration curves for each set of primers were between 83– GGTGGCTTCC. Internal sequences were selected for the P3H3 93%. Fold differences in target genes were normalized either to probe where the gene was most divergent from P3H1 and P3H2 GAPDH expression or 18srRNA expression in each tissue where and these amplified a 274 bp product by PCR. The P3H3 gene indicated (ΔΔCT method). Additionally gene specific data were specific oligonucleotides selected were: 5'GACACTGGATACG- quantified using the standard curves for each gene and normalized GGTTCTG and 5'ATGTCCTCTCGTGGTCCCAG. The GAPDH to total input RNA (ΔCT method). Data analysis was performed oligonucleotide sequences selected amplified a 150 bp product by using the iQ5 Optical System Software version 2.0. PCR and were as follows: 5'CACTGCCACCCAGAAGACTGT and 5'GGAAGGCCATGCCAGTGA. The PCR products were Whole mount in situ hybridization and cryosection labeled using a kit for random priming (Roche Random Primed analysis DNA Labeling Kit) and [α32P]dCTP. Internal probes of approximately 600 nucleotides in length were E10–E15 embryos were fixed overnight in 4% paraformaldehyde also generated to P3H1 and P3H2 internal sequences for each in 1x phosphate buffered saline (PBS) at 4°C, then dehydrated in gene. The following oligonucleotides were used to PCR-amplify methanol and stored at –20°C until use. Hybridization was carried the gene-specific products: 5'GCTGGCTTCCCACCCAAGTA out overnight at 70°C as described previously with the addition of and 5'GCAGCTCCTGGCACTCATCA for the P3H1 probe and the digioxigenin-labeled P3H1, P3H2, and P3H3 RNA probes at 5'CCAAGCCACACCTGGAAAGC and 5'CCTGCCGTCCTCCA- 0.5 μg/ml (Carpenter et al., 1993; Manley and Capecchi, 1995; TAGCTG for the P3H2 probe. Morgan et al., 2003). Alkaline phosphatase-conjugated anti- digoxigenin Fab fragments were used at 1:3000 (Roche) as describ- Real time quantitative PCR ed by manufacturer. Color reactions were carried out over time periods ranging from 2 hours to overnight for optimized signal. Total mouse RNA was purchased from BD Biosciences (Mouse For the antisense probes the same oligonucleotides as described Total RNA Master Panel). Each representative RNA (from adult in the methods above were used to PCR-amplify 600 bp gene- mouse heart, brain, eye, lung, kidney, liver, uterus, testis, spleen, specific 3'UTR products. These were subsequently cloned into the thymus, salivary gland, and total RNA from embryonic stages E7, pCR4 cloning vector (Invitrogen) which contains RNA Polymerase E11, E15 and E17) was reverse transcribed using Superscript T3 and T7 promoters. Antisense riboprobes were generated using Reverse Transcriptase III (Invitrogen) according to the manufac- the plasmids and digoxigenin UTP and T7 or T3 RNA polymerase

98 P3H Gene Expression in Mice at 37°C for 2 hours according to manufacturer’s instructions (Roche). For cryosection analysis, paraformaldehyde-fixed embryos were washed 30 min in 1x PBS. Cryoprotection of the tissues was achieved using a sequential series of 10–40% sucrose in 1x PBS. The embryos were oriented in OCT (Tissue-Tek) and frozen rapidly on dry ice. The embryos were sectioned to 16 μm using a Leitz Kryostat 1740, and mounted on Superfrost plus slides (Fish- er) for analysis. Sections were rinsed for 10 min three times in 1x PBS to remove the OCT. They were then treated with 1 μg/ml pro- teinase K for 5 min at room temperature, washed in PBS with 0.1% Tween 20 (PBST) and fixed in 4% paraformaldehyde. Sections were washed in PBST and acetylated in acetic anhydride and 0.1 M triethanolamine for 15 min at room temperature. Slides were washed in PBST and then air dried followed by the addition of pre- warmed hybridization solution and incubation at 65°C for 1 hour in humidified boxes. Antisense riboprobes were added to slides at 0.5 μg/ml and incubated overnight at 65°C. Sections were washed in 1x SSC/50% formamide for 30 min at 65°C and then treated with 20 μg/ml RNase A (Roche) for 30 min at 37°C. Slides were then washed in 2x SSC, and twice in 0.2x SSC each for 20 min at 65°C. Sections were then blocked in 20% heat-inactivated sheep serum (HISS) for 1 hour at room temperature and then incubated in a 1:2500 dilution of anti-digoxigenin-AP (Roche) in 5% HISS overnight at 4°C. Slides were washed four times in 0.1 M maleic acid, 0.15 M NaCl for 5 min each wash and then developed with 250 μg/ml NBT and 125 μg/ml BCIP in 0.1 M NaCl, 0.1 M Tris, pH 9.5, and 0.05 M MgCl2 overnight at 4°C.

Results

Northern blot analysis of P3H1, P3H2 and P3H3 expression in mouse tissues Northern blot analysis was performed on various adult mouse tissues to determine transcript size using probes derived from the 3'UTR for all three P3H genes (Fig. 1A– C). Two transcripts of approximately 2.8 and 3.5 kb were Fig. 1. Northern blot analysis of RNA from adult mouse tissues. detected for P3H1 in heart, liver, and kidney (Fig. 1A). The Hybridizations were performed using radiolabeled 3'UTR probes to detect prolyl 3-hydroxylase 1 (A) transcripts, prolyl 3-hydroxylase 2 (B) two different transcripts are thought to represent a long transcripts, and prolyl 3-hydroxylase 3 (C) transcripts. RNA from adult form and an alternatively spliced shorter form, both of mouse tissues were loaded in the following lanes: 1. Heart, 2. Brain, 3. which have previously been found to be associated with cer- Liver, 4. Skeletal Muscle, 5. Kidney. P3H1 transcripts were detected at tain human tissues and transformed cells (Kaul et al., 2000). approximately 2.8 and 3.5 kb (A). P3H2 transcripts were detected at P3H1 expression was not detected in skeletal muscle by approximately 3.7 kb in most tissues with an additional 2.0 kb transcript in northern blot analysis with either the 3'UTR probe or with liver (B). P3H3 transcripts were detected approximately as a 3.0 kb an internal probe (see Methods). When the blot was hybrid- transcript in heart, lung, liver and kidney, as well as a 3.8 kb transcript in brain (C). GAPDH was detected in all tissues as a 1.3 kb transcript and ized with a GAPDH probe (Fig. 1D) the presence of a single verifies the integrity of the RNA on the blots (D). 1.3 kb transcript in all lanes indicated that RNA was present for all samples on the blot; however, GAPDH expression appeared to vary significantly from tissue to tissue (see ous studies of human P3H2 expression reported an addi- Discussion.) Prolyl 3-hydroxylase 2 (P3H2) was detected tional 6.5 kb transcript in skeletal muscle (Jarnum et al., approximately as a 3.7 kb transcript in kidney, heart, and 2004). Finally, prolyl 3-hydroxylase 3 (P3H3) was detected liver, as well as a 2.0 kb transcript only found in liver (Fig. as a 3.0 kb transcript in heart, liver, and kidney (Fig. 1C). A 1B). It is not clear whether the smaller transcript in liver 3.8 kb P3H3 transcript was also detected in the brain represents a splice variant or a degradation product. Previ- whereas neither P3H1 nor P3H2 was detected in this same

99 J. Vranka et al. tissue. Differences in transcript size may be due to alterna- Table I. EXPRESSION OF PROLYL 3-HYDROXYLASE GENES AT DIFFERENT tively processed transcripts and may result in tissue-specific DEVELOPMENTAL STAGES NORMALIZED TO TOTAL INPUT RNAa differences of overall enzyme activity as the enzymatic Mouse Tissue P3H1 P3H2 P3H3 activity resides in the carboxy-terminal domain of all three E7b 1.07±0.12c 0.37±0.04 0.09±.008 proteins. It is important to note here that although northern blots are a good method to determine correct transcript size, E11 0.56±0.04 1.16±0.28 0.94±0.09 quantitative PCR is the primary method used in this work E15 1.16±0.17 2.04±0.27 1.47±0.16 to assess actual quantitative differences in P3H expression E17 0.63±0.10 0.94±0.24 0.39±0.03 levels across various tissue samples. a Total input RNA=100 ng per PCR reaction. b E represents embryonic day. c Error bars are standard deviation values from three different experiments. Real-time quantitative PCR analysis of P3H1, P3H2, and P3H3 in mouse tissues Table II. EXPRESSION OF PROLYL 3-HYDROXYLASE GENES IN ADULT Real-time quantitative PCR on reverse-transcribed RNA MOUSE TISSUES NORMALIZED TO TOTAL INPUT RNAa from a variety of adult mouse tissues, as well as from Mouse Tissue P3H1 P3H2 P3H3 embryonic mice at developmental stages (E7–E17) was per- b formed to quantitate the expression levels of P3H1, P3H2, Brain 0.23±0.02 0.45±0.06 0.44±0.06 and P3H3 genes (Table I and Table II). The most striking Eye 0.14±0.01 1.13±0.18 0.19±0.04 difference in gene expression was a tenfold higher expres- Heart 0.36±0.07 1.10±0.33 0.21±0.02 sion of P3H2 in the adult kidney, a tissue rich in basement Kidney 0.22±0.03 12.7±3.10 0.08±.007 membrane type IV collagen which is a likely substrate of Liver 0.23±0.01 0.13±.006 0.03±.002 the P3H2 enzyme (Table II and Fig. 2A). P3H2 was also Submax. Gland 0.03±.003 0.02±0.01 0.05±.002 expressed at higher levels in the heart, spleen, and eye Uterus 0.24±0.04 0.41±0.07 0.21±0.02 (Table II and Fig. 2A). Slight differences in expression Testis 0.03±.001 0.12±.009 0.03±.004 levels of P3H2 between Fig. 2 and Table II are likely due to Spleen 0.06±.009 0.91±0.10 0.07±.007 the normalization of expression levels to either 18srRNA or Thymus 0.08±.002 0.16±.009 0.06±0.01 to total input RNA, respectively. Interestingly, collagen IV Lung 0.19±0.03 0.56±0.05 0.17±.009 is an essential component of the basement membranes in Bone 0.10±.007 0.15±.007 0.04±.010 kidney, spleen and the lens of the eye (Maatta et al., 2004; Cartilage 0.22±0.02 0.12±0.01 0.12±0.04 Pöschl et al., 2004). Skin 0.12±.002 0.27±0.10 0.05±0.01 For P3H3 the highest levels of expression were detected a Total input RNA=100 ng per PCR reaction. in brain, heart, and uterus. P3H1 was expressed at low b Error bars are standard deviation values from three different experiments. levels in heart, liver, and kidney and at slightly higher levels in cartilage relative to P3H2 or P3H3 (Table II and Fig. 2). All three genes were expressed at low levels in adult tion which relates the mRNA data to the total RNA content thymus, testis, submaxillary gland and bone (Fig. 2A, 2B of the sample preparation subjected to the reverse tran- and Table II). scription reaction, (Bustin, 2002; Bustin and Nolan, 2004; Differences in the embryonic expression of P3H1, P3H2 Sindelka et al., 2006). Very low levels of expression of any and P3H3 were also detected. For P3H1, embryonic expres- of the 3 prolyl 3-hydroxylase genes in the tissues presented sion appeared highest at approximately equal levels at both in Table II are not thought to be significant. E7 and E15 (Table I and Fig. 2B). P3H2 and P3H3 were also expressed but at the mid-embryonic stages E11 and In situ hybridization on embryonic mouse tissues using E15 (Table I and Fig. 2B). These results demonstrate some 3'UTR antisense riboprobes overlap in the expression of the three genes as well as some distinct expression differences in specific tissues supporting Our previous characterization of P3H1 in the developing the idea that the enzymes may be important in development chick suggested that tissues containing fibrillar collagens and have some unique tissue specificities. Some small dis- such as cartilage, tendon, bone, skin, and aorta were likely crepancies in the expression levels of a gene are present in targets of P3H1 enzymatic activity (Vranka et al., 2004). In the data and are thought to be due to the method of normal- this study embryonic mouse tissues were analyzed by whole ization as well as the housekeeping gene used, as repre- mount and section in situ hybridization using riboprobes sented in Fig. 2 (fold expression relative to 18srRNA) specific for P3H1, P3H2 and P3H3 to determine whether versus in Table II (expression normalization to total input there was a similar pattern of P3H distribution. Common RNA). As mRNA expression of reference genes has been and unique sites of expression of P3H1, P3H2 and P3H3 shown to differ among tissues and various cells the P3H were detected in the embryos throughout the developmental gene levels were analyzed by the method of data normaliza- stages analyzed (E11.5–E13.5). Among the regions express-

100 P3H Gene Expression in Mice

Fig. 2. Graphical representation of real-time quantitative PCR analysis of adult and embryonic mouse tissues. Data are represented as the fold change differences and are represented as the ratio of target gene expression to 18srRNA signal (A, B) per 100 ng of input RNA from each tissue. All experiments were performed in triplicate and mean values are represented on each graph. P3H1 gene expression is represented by the black boxes, P3H2 is in white and P3H3 is in gray. Graph A shows relative gene expression in adult tissues. Graph B shows relative P3H gene expression in embryonic mouse tissues.

ing all 3 prolyl 3-hydroxylase genes were the forelimbs and Discussion hindlimbs, and maxillary and mandibular components of the first embryonic arch at E11.5 (Fig. 3A–C), in the distal tip A developmental role for P3H1 is most likely linked to that of the tail and in the vibrissae at E12.5 (Fig. 3D–F), and in of the fibrillar collagens, particularly types I, II, and III the developing pinnae at E13.5 (Fig. 3G–I). P3H2 and which are abundantly expressed during development P3H3 were also expressed in the developing vertebral bod- (Niederreither et al., 1995). Mutations in P3H1 have been ies (Fig. 3E and 3F) and in the developing tail (Fig. 3G–I). shown to cause a recessive form of osteogenesis imperfecta P3H3 expression was also detected in the hair follicles of demonstrating a crucial role for P3H1 in facilitating col- the skin at E13.5 (Fig. 3I). lagen stability and bone development (Cabral et al., 2007). Finally to identify the cell types expressing the P3H Additionally, type II collagen is expressed in chondrogenic genes in situ hybridization was performed on E12.5 cryo- tissue in advance of chondrogenesis, as well as in some non- sections (Fig. 4). P3H1 was expressed in prechondrogenic chondrogenic tissues of the developing mouse embryo skeletal elements, such as the vertebral bodies (Fig. 4A) and including the heart, epidermis, inner ear, and nervous sys- cranial primordia (Fig. 4C). P3H1 was also strongly tem (Cheah et al., 1991; Ng et al., 1993; Sakai et al., 2001) expressed in the aortic arch (Fig. 4B). In contrast, P3H2 was indicating the involvement of prolyl 3-hydroyxlase enzyme not expressed in precartilaginous structures (Fig. 4D) or in activity during development. Collagen IV has also been the aortic arch (Fig. 4E). P3H2 expression was observed in shown to be indispensable for the structural integrity of the cells forming part of the intervertebral discs (Fig. 4D) basement membranes at later stages of development and in and in the lens of the eye (Fig. 4F), whereas P3H1 expres- adult tissues (Pöschl et al., 2004). A recent study demon- sion was not detected in the eye at E12.5 (Fig. 4C). P3H3 strated that P3H1 protein was present in the stromal was also expressed in the eye in subpopulations of cells elements of the rat kidney during development with its within the lens (Fig. 4I). Overall, P3H3 appeared to be more expression persisting into adulthood (Lauer et al., 2007). ubiquitously expressed throughout the E 12.5 mouse embryo This data is in agreement with our gene expression data in (Fig. 4G–I) and showed some overlap with P3H1 expres- which P3H1 is present in kidney but at limited levels. The sion in the vertebral bodies (Fig. 4A–C) and with P3H2 highly elevated level of P3H2 expression in the adult kidney expression in the eye (Fig. 4D–F). supports the idea that its enzymatic activity is essential to the development and formation of functional basement membranes. These results are supported by the recent work of Tiainen et al. showing P3H2 expression in fetal kidney (Tiainen et al., 2008). Additionally, a very recent study has

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Fig. 3. Detection of prolyl 3-hydroxylase gene expression in whole mouse embryos by in situ hybridization. Antisense riboprobes were generated to a 600 bp fragment of the 3'UTR region of P3H1 (A, D, G), P3H2 (B, E, H), and P3H3 (C, F, I) and used to detect gene expression levels. P3H1 detected in the forelimb and hindlimb at E11.5 (A), E12.5 (D), and E13.5 (G). P3H2 detected in the forelimb and hindlimb of E11.5 embryos (B), E12.5 (E) and also in the distal tip of the tail (asterisks) (E), and limbs of the E13.5 embryo (H). P3H3 detected in the forelimb and hindlimb at stages E11.5 (C), E12.5 (F), and E13.5 (I) as well as in the distal tip of the tail (asterisks), vertebral bodies (arrows), vibrissae (v), and hair follicles in the skin (arrowheads) (I). fl=forelimb; hl=hindlimb, p=pinnae, t=tail.

demonstrated not only conserved notochord expression of In summary we conclude from the data presented here all three prolyl 3-hydroxylase genes but also the co- that the prolyl 3-hydroxylases have both unique and over- expression of the three genes in areas of the mouse embryos lapping regions of expression in adult and embryonic tis- at various developmental stages suggesting a functional sues. This differential expression may reflect the utilization importance for the development of specific regions of these proteins in their abilities to act upon a unique set of (Capellini et al., 2008). More detailed studies on the expres- substrates in discrete tissues. P3H1 appears to be coordi- sion of P3H3 in a wider variety of tissues, cell types, and nately expressed and to recognize fibrillar collagens such as ages will help elucidate its potential substrates and cellular types I, II, III, and V collagen in adult and developing tis- activities. sues. P3H2 appears to be coordinately expressed with and

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Fig. 4. Detection of prolyl 3-hydroxylase gene expression in E12.5 mouse tissue cryosections by in situ hybridization. Antisense riboprobes made to P3H1 (A–C), P3H2 (D–F), and P3H3 (G–I) were used to detect expression levels in the cartilage condensations of the developing vertebrae (arrowheads) (A, D, G), the aortic arch (arrows) and Meckel’s cartilage (B, E, H), and the developing eye (C, F, I). P3H1 is detected in cartilage condensations in the vertebrae (A), whereas P3H2 localizes to intervertebral zone (D), and P3H3 is detected throughout the entire vertebral area (G). P3H1 is detected within the aortic arch (arrows) and in Meckel’s cartilage (B), whereas P3H2 (E) and P3H3 (H) are not localized to either tissue. P3H1 is detected in the developing mouse eye in cartilage primordium well below the retina (C), however P3H2 is localized to the lens of the eye (F) and P3H3 is detected in the retina, cornea, and within lens cell subpopulations (I). iv=intervertebral zone; lg=lung; mc=Meckel’s cartilage; aa=aortic arch; c=cornea; l=lens; r=retina; c.p.=cartilage primordium. therefore recognizes collagens such as References type IV collagen, which is known to contain up to 15 3-(S)- Barnes, A.M., Chang, W., Morello, R. et al. 2006. Deficiency of cartilage- hydroxyproline residues in 1000. Other basement mem- associated protein in recessive lethal osteogenesis imperfecta. N. Engl. J. brane associated collagens may be targets of this enzyme as Med., 355: 2757–2764. well. The potential substrates of the third P3H family mem- Bustin, S.A. 2002. Quantification of mRNA using real-time reverse tran- ber are ambiguous, but may be collagens or other proteins scription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol., 29: 23–39. predominantly expressed in brain or uterus and expressed in Bustin, S.A. and Nolan, T. 2004. Pitfalls of quantitative real-time reverse- some developing mouse tissues. The data presented herein transcription polymerase chain reaction. J. Biomol. Tech., 15: 155–166. help to further the current understanding of the family of Cabral, W.A., Chang, W., Barnes, A.M. et al. 2007. Prolyl 3-hydroxylase prolyl 3-hydroxylase genes as well as to suggest potentially 1 deficiency causes a recessive metabolic bone disorder resembling novel individual enzyme substrates. lethal/severe osteogenesis imperfecta. Nat. Genet., 39: 359–365. Capellini, T.D., Dunn, M.P., Passamaneck, Y.J., Selleri, L., and Di Gregorio, A. 2008. Conservation of notochord gene expression across Acknowledgements. This study was supported in part by a Shriners Hospital chordates: insights from the gene family. Genesis, 46: 683– Postdoctoral Research Fellowship (J. Vranka) and grants from Shriners 696. Hospital for Children Research (H.P.B. & H.S.S.) Carpenter, E.M., Goddard, J.M., Chisaka, O., Manley, N.R., and Capecchi, M.R. 1993. Loss of Hox-A1 (Hox-1.6) function results in the reorgani-

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zation of the murine hindbrain. Development, 118: 1063–1075. cysteine-rich amino-propeptide in differentiating cartilage and Cheah, K.S., Lau, E.T., Au, P.K., and Tam, P.P. 1991. Expression of the nonchondrogenic tissues during early mouse development. Dev. Biol., mouse alpha 1(II) collagen gene is not restricted to cartilage during 159: 403–417. development. Development, 111: 945–953. Niederreither, K., D’Souza, R., Metsaranta, M., Eberspaecher, H., Toman, Fietzek, P.P., Rexrodt, F.W., Hopper, K.E., and Kühn, K. 1973. The P.D., Vuorio, E., and De Crombrugghe, B. 1995. Coordinate patterns of covalent structure of collagen. 2. The amino-acid sequence of alpha1- expression of type I and III collagens during mouse development. Matrix CB7 from calf-skin collagen. Eur. J. Biochem., 38: 396–400. Biol., 14: 705–713. Fietzek, P.P., Wendt, P., Kell, I., and Kühn, K. 1972. The covalent struc- Pöschl, E., Schlotzer-Schrehardt, U., Brachvogel, B., Saito, K., Ninomiya, ture of collagen: amino acid sequence of 1-CB3 from calf skin collagen. Y., and Mayer, U. 2004. Collagen IV is essential for basement mem- FEBS Lett., 26: 74–76. brane stability but dispensable for initiation of its assembly during early Jarnum, S., Kjellman, C., Darabi, A., Nilsson, I., Edvardsen, K., and development. Development, 131: 1619–1628. Aman, P. 2004. LEPREL1, a novel ER and Golgi resident member of Rexrodt, F.W., Fietzek, P.P., and Kühn, K. 1975. The covalent structure the Leprecan family. Biochem. Biophys. Res. Commun., 317: 342–351. of collagen. The chymotrypsin, trypsin and hydroxylamine peptides Kaul, S.C., Sugihara, T., Yoshida, A., Nomura, H., and Wadhwa, R. 2000. derived from alpha2-CB4 of calf-skin collagen. Eur. J. Biochem., 59: Gros1, a potential growth suppressor on chromosome 1: its identity to 105–112. basement membrane-associated , leprecan. Oncogene, 19: Rhodes, R.K. and Miller, E.J. 1978. Physicochemical characterization and 3576–3583. molecular organization of the collagen A and B chains. Biochemistry, Kefalides, N.A. 1972. Isolation and characterization of cyanogen bromide 17: 3442–3448. peptides from basement membrane collagen. Biochem. Biophys. Res. Sakai, K., Hiripi, L., Glumoff, V., Brandau, O., Eerola, R., Vuorio, E., Commun., 47: 1151–1158. Bösze, Z., Fässler, R., and Aszódi, A. 2001. Stage-and tissue-specific Kefalides, N.A. 1973. Structure and biosynthesis of basement membranes. expression of a Col2a1-Cre fusion gene in transgenic mice. Matrix Biol., Int. Rev. Connect. Tissue Res., 6: 63–104. 19: 761–767. Lauer, M., Scruggs, B., Chen, S., Wassenhove-McCarthy, D., and McCarthy, Sindelka, R., Ferjentsik, Z., and Jonak, J. 2006. Developmental expression K.J. 2007. Leprecan distribution in the developing and adult kidney. profiles of Xenopus laevis reference genes. Dev. Dyn., 235: 754–758. Kidney Int., 72: 82–91. Tiainen, P., Pasanen, A., Sormunen, R., and Myllyharju, J. 2008. Charac- Maatta, M., Liakka, A., Salo, S., Tasanen, K., Bruckner-Tuderman, L., terization of recombinant human prolyl 3-hydroxylase isoenzyme 2, an and Autio-Harmainen, H. 2004. Differential expression of basement enzyme modifying the basement membrane collagen IV. J. Biol. Chem., membrane components in lymphatic tissues. J. Histochem. Cytochem., 283: 19432–19439. 52: 1073–1081. Tonachini, L., Morello, R., Monticone, M., Skaug, J., Scherer, S.W., Manley, N.R. and Capecchi, M.R. 1995. The role of Hoxa-3 in mouse Cancedda, R., and Castagnola, P. 1999. cDNA cloning, characterization thymus and thyroid development. Development, 121: 1989–2003. and chromosome mapping of the gene encoding human cartilage Morello, R., Bertin, T.K., Chen, Y. et al. 2006. CRTAP is required for associated protein (CRTAP). Cytogenet. Cell Genet., 87: 191–194. prolyl 3-hydroxylation and mutations cause recessive osteogenesis Vranka, J.A., Sakai, L.Y., and Bächinger, H.P. 2004. Prolyl 3-hydroxylase imperfecta. Cell, 127: 291–304. 1, enzyme characterization and identification of a novel family of Morello, R., Tonachini, L., Monticone, M., Viggiano, L., Rocchi, M., enzymes. J. Biol. Chem., 279: 23615–23621. Cancedda, R., and Castagnola, P. 1999. cDNA cloning, characterization Willaert, A., Malfait, F., Symoens, S., Gevaert, K., Kayserili, H., and chromosome mapping of Crtap encoding the mouse cartilage associ- Megarbane, A., Mortier, G., Leroy, J.G., Coucke, P.J., and De Paepe, A. ated protein. Matrix Biol., 18: 319–324. 2008. Recessive Osteogenesis Imperfecta caused by LEPRE1 muta- Morgan, E.A., Nguyen, S.B., Scott, V., and Stadler, H.S. 2003. Loss of tions: clinical documentation and identification of the splice form Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia. responsible for prolyl 3-hydroxylation. J. Med. Genet., 46: 233–241. Development, 130: 3095–3109. Ng, L.J., Tam, P.P., and Cheah, K.S. 1993. Preferential expression of (Received for publication, March 18, 2009, accepted, July 25, 2009 and alternatively spliced mRNAs encoding type II procollagen with a published online, August 1, 2009)

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