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Collagen prolyl 4-hydroxylase is up-regulated in an acute bladder outlet obstruction
Journal of Pediatric Urology (2006) 2, 225e232
Collagen prolyl 4-hydroxylase is up-regulated in an acute bladder outlet obstruction Sang Don Lee b, Cem Akbal a, Rosalia Miseeri a, Chaeyong Jung a, Richard Rink a, Martin Kaefer a,* a
Department of Urology, Riley Hospital for Children, Indiana University, 702 Barnhill Drive, Indianapolis, IN 46202, USA b College of Medicine, Pusan National University, Busan, South Korea Received 26 September 2005; accepted 21 March 2006 Available online 7 July 2006
KEYWORDS Prolyl 4-hydroxylase; Bladder smooth muscle; Bladder outlet obstruction
Abstract Objective: Compliance is primarily related to extracellular matrix deposition, and prolyl 4-hydroxylase (P4Hs) plays a critical role in the synthesis of the matrix. To study the alteration of P4Hs, under the influence of variable hydrostatic pressure, a novel pressure device was used to expose human bladder smooth muscle cells (HBSMC) and fibroblasts (HBF) to pressures in the physiologic range. We then studied acute obstructed porcine bladder tissues to see if changes can also be seen after in-vitro obstruction. Materials and methods: HBSMC and HBF were exposed to pressures at 0, 20 and 40 cmH2O for up to 72 h. In-vivo studies were carried out next, using six normal (control) and five obstructed porcine bladders. Pigs were exposed to a consistent hydrostatic pressure of 20 cm for 24 h after ligation of the urethra. We used 2-DE to compare protein profiling of HBSMC under normal and increased pressures. Other analyses were used to detect molecular alteration and altered expression of mRNA for P4Hs. Results: We identified 437 proteins from 476 spots (91.8%) obtained from HBSMC that were differentially expressed under normal and increased pressures. Under increased pressure, 48 unique proteins were significantly increased or decreased, and a prominent protein regulating extracellular matrix synthesis highly correlated with P4Hs. The exposure of both HBSMC and HBF to a sustained hydrostatic pressure resulted in the increased expression of P4Hs in a time- and pressure-dependent manner. In vivo, P4Hs expression was also significantly increased in the obstructed group. Conclusions: P4Hs is up-regulated, in the human bladder, time and pressure dependently. The alteration of P4Hs over a short period may significantly influence the synthesis of extracellular matrix in vivo and lead to decreased compliance.
* Correspondence author. Tel.: þ1 317 274 8896; fax: þ1 317 274 7481. E-mail address: [email protected] (M. Kaefer). 1477-5131/$30 ª 2006 Published by Elsevier Ltd on behalf of Journal of Pediatric Urology Company. doi:10.1016/j.jpurol.2006.03.011
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S.D. Lee et al. Our results also support the concept that bladder outlet obstruction, with resultant pressures of 40 cmH 2 O or less, results in molecular changes consistent with decreased compliance. ª 2006 Published by Elsevier Ltd on behalf of Journal of Pediatric Urology Company.
Introduction Bladder outlet obstruction (BOO) with intravesical pressures exceeding 40 cmH2O results in alterations of bladder physiology, including wall thickening, reduced compliance and decreased capacity. From a biomechanical standpoint, compliance is primarily related to extracellular matrix deposition. Information, at the level of the proteome, is necessary to unravel the critical changes in extracellular matrix deposition involved in the pathogenesis of BOO. Comparative studies of protein expression, under normal and pathologic conditions, have led to the identification of aberrantly expressed proteins that may represent important new markers [1]. Two-dimensional gel electrophoresis (2-DE) is a powerful research technique that makes possible simultaneous examination of hundreds of polypeptides in a tissue or cell sample [2]. To quantify the proteins regulating the production of extracellular matrix under normal and pathologic conditions, 2-DE was performed in human bladder smooth muscle cells (HBSMC). Among several individual HBSMC protein spots, a prominent spot for the production of extracellular matrix highly correlated with prolyl 4-hydroxylase (P4Hs). BOO causes overproduction of extracellular matrix containing collagen. Synthesis of collagen, the major component of the extracellular matrix, begins with the translocation of collagen messenger RNA (mRNA) on the ribosomes of the rough endoplasmic reticulum, and hydroxylation of its proline moiety to hydroxyproline by P4Hs. Proline hydroxylation is thought to stabilize the triple helix, Xaa-Pro-Gly, in the collagen molecule, and P4Hs is believed to be a key enzyme in the regulation of collagen synthesis [3e5]. To date there have been no reports on whether a mechanical stimulus, such as hydrostatic pressure, has an effect on P4Hs. Furthermore, the threshold pressure, above which alterations in molecular key enzymes for bladder compliance occur, has not been determined. To study the alteration of P4Hs, under the influence of variable hydrostatic pressures, we employed a novel pressure device [6] to expose HBSMC and human bladder fibroblasts (HBF) to
pressures in the physiologic range. Acute obstructed porcine bladder tissues were subsequently evaluated to see if these changes were also seen as a result of in vitro obstruction.
Materials and methods Human bladder smooth muscle cells (HBSMC) and fibroblasts Human bladder smooth muscle cells (HBSMC) (Clonetics, East Rutherford, NJ, USA) and Human bladder fibroblast (HBF) were expanded in culture using smooth muscle cell basal media (SmBM, Clonetics) containing fetal bovine serum (5%), insulin (5 mg/ml), human recombinant fibroblast growth factor-b (2 ng/ml), human recombinant epidermal growth factor (0.5 ng/ml), gentamycin (15 mg/ml) and amphotericin (7.5 ng/ml) according to the manufacturer’s protocol. These cells were plated on P100 culture dishes and cultured for 2 days under standard cell-culture conditions (a sterile, humidified, 37 C, 95% air/5% CO2 environment). Thereafter, the media was changed to serum-free Leibovitz media (Invitrogen) and cells were placed in a pressure chamber under pressures of 20 and 40 cmH2O for 24, 48 and 72 h.
Computer-operated pressure system To expose the cultured HBSMC and HBF to a controlled stimulus of increased hydrostatic pressure, we used a custom-made, computer-operated pressure system, previously described by Backhaus et al. [6]. Briefly, a computer with software specially developed for this system controlled and maintained a designated pressure environment inside a sealed, acrylic chamber that housed standard tissue-culture plates of HBSMC and HBF. A computer interface controlled pressure levels, maintained (with feedback adjustments) the desired pressure for the duration of the experiments and collected pressure data. The prescribed pressure inside the chamber was monitored and maintained using a pressure transducer, and inlet and outlet solenoid valves that controlled the flow of compressed air mixture into and out of the system.
P4Hs is up-regulated in an acute bladder obstruction During experiments with HBSMC and HBF, the pressure chamber was placed in an incubator without involvement of CO2, and cells were kept in Leibovitz’s media designed for growth of cells in a CO2-free environment. This set up eliminates effects of pH change.
Experimental animals Following approval from the Institutional Animal Care and Use Committee (IACUC), the in vivo study was carried out using male pigs weighing an average of 29.8 kg. The animals were maintained in pathogen-free animal facilities according to IACUC guidelines.
227 the tissues, protein fractions were prepared by grinding the frozen tissue samples in a mortar and pestle. The pulverized tissues were homogenized with Poly Tron PT-MR2100 (Kinematica AG, Littau-Luzem, Switzerland) in modified RIPA buffer. The homogenates were centrifuged for 10 min at 14,000 g, 4 C. The pellet was resuspended and re-homogenized in modified RIPA buffer with 5% Triton X, and then incubated for 1 h on ice. The final homogenates were centrifuged for 2 min at 14,000 g, 4 C, and the supernatant (solubilized protein) was isolated and stored at 80 C until needed.
Two-dimensional gel electrophoresis and protein quantification
Surgical procedure The pigs were anesthetized using inhalation halothane, and a left paramedian incision was made in the lower abdomen. The urethra was exposed, and a No.1 silk suture was tied around the urethra. A new bladder outlet was created by the placement of a supra-pubic catheter into the bladder; into the lumen of this catheter a valve was placed that allowed urine to flow out of the bladder when a pre-determined pressure differential was generated. Valves with opening resistances of 20 cmH2O were used. Implantable, radiotelemetered manometry devices were placed into the bladder and abdomen to provide realtime ambulatory urodynamic assessment. The incision was closed with surgical sutures, and the pigs were housed in separate cages for 24 h. After 24 h of bladder outlet obstruction the pigs were killed. The body of the urinary bladder (tissue above the ureteral orifice) was dissected free of the serosa and surrounding fat. The bladder was divided in the midsagittal plane, and then cut into longitudinal smooth muscle strips (w5 20 mm). The muscle strips were rapidly frozen and stored at 80 C until needed.
Preparation of protein extracts Following exposure of cultured cells (HBSMC and HBF) to the hydrostatic pressure stimulus, the culture medium was removed, and cells were washed with 10 ml of ice-cold phosphate-buffered saline (PBS, pH 7.4) and lysed in modified RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, plus proteinase inhibitors). The cells were then scraped off the plates, transferred to a centrifuge tube, and centrifuged at 14,000 g, 4 C for 10 min. For
Sample preparation Harvested HBSMC were disrupted with a cocktail of 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris (hydroxymethyl) aminomethane, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The resulting cell lysates were subjected to physical shearing by passing through a syringe fitted with a 21-G needle, followed by syringes with 25-G and 27-G needles successively, and the addition of 50 mg/mL DNase I and 50 mg/mL RNase A. The sample was then centrifuged using a Beckman TL-100 Tabletop Ultracentrifuge (Palo Alto, CA) at 85,000 rpm for 2 h at 15 C. Two-dimensional gel electrophoresis The first dimension isoelectric focusing (IEF) was performed on precast 18-cm immobilized pH gradient (IPG) strips (Amersham Pharmacia Biotech) at 15 C with a maximum current setting of 50 mA/strip using an Amersham Pharmacia IPGphor IEF unit. The strips were rehydrated for a minimum of 10 h in ceramic strip holders in 350 mL of sample containing 7 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF, 20 mM dithiothreitol (DTT), and 0.5% IPG buffer (Amersham Pharmacia Biotech). The amount of protein loaded was w150 g for analytical gels and w350 mg protein for preparative gels. A low voltage of 30 V was applied during rehydration. After rehydration, the IEF run was carried out under the following conditions: 500 V/500 V h, 1000 V/1000 V h, and 8000 V/32000 V h. Voltage increases were performed on a step-wise basis. Before carrying out the second-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the strips were subjected to a two-step equilibration. The first was with an equilibration buffer
228 consisting of 6 M urea, 30% glycerol, 2% SDS, 50 mM TriseHCl (pH 6.8), 1% w/v DTT. The second step was with a buffer consisting of 6 M urea, 30% glycerol, 2% SDS, 50 mM TriseHCl (pH 8.8), 2.5% w/v iodoacetamide (Sigma). After the IPG strips were transferred onto the second-dimensional SDS-PAGE gel, the strips were sealed in place with 0.75% agarose. SDS-PAGE was performed on 1.0-mm thick 10% polyacrylamide gels at a constant voltage of 110 V at 10 C using an Amersham Pharmacia Iso-Dalt electrophoresis unit. Protein digestion and MALDI-MS analysis Protein identification by peptide mass fingerprinting [7] was carried out as described in detail previously [8]. Briefly, altered proteins were excised from replicate gels and digested with trypsin. The masses of the resulting peptides were determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Database searching and identification of proteins The proteins were identified by searching in SWISSPROT and NCBI non-redundant databases using MS-Fit (Protein prospector, UCSF, San Francisco, CA, USA). All mass searches were performed using a mass window between 1000 and 100,000 Da. The search parameters allowed for oxidation of methionine, N-terminal acetylation, carboxyamidomethylation of cysteins, and phosphorylation of serine, threonine and tyrosine. The criteria for positive identification of proteins were set as follows: at least four matching peptide masses and 50 ppm or better mass accuracy, and molecular weight and isoelectric point (pI) of identified proteins were required to match estimated values obtained from the image analysis.
Western blot analysis To determine P4Hs expression, the membrane proteins extracted from whole-cell lysates and bladder tissues were loaded onto 4e12% Novex Trise glycine electrophoresis gels (Invitrogen) for separation at 125 V, before blotting onto a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Membranes were blocked with 5% nonfat milk power in PBS at 25 C for 1 h before incubation for 2 h at 25 C with mouse antihuman-prolyl 4 hydroxylase (b-subunit) monoclonal antibody (Chemicon) at a dilution of 1:1000. After washing the membrane, a further incubation for 1 h at 25 C with anti-mouse horseradish
S.D. Lee et al. peroxidase-conjugated secondary antibody (Pierce) was performed before visualization using chemiluminescence substrate (Pierce).
Reverse transcription-polymerase chain reaction (RT-PCR) To determine the expression of mRNA for P4Hs, total RNA was extracted from porcine bladder tissues, HBSMC and HBF using TRIZOL solution (Invitrogen) according to the manufacturer’s protocol. RT was performed in a total reaction volume of 20 ml using 1 mg of total RNA, 1 ml Supercript II (Moloney’s murine leukemia virus reverse transcriptase, Invitrogen) and 100 pmol of oligo (dT)15 as a first-strand primer at 42 C for 1 h. PCR was performed using primers (sense 50 -GAA ACG GAT AAT ACG AGG CTT GTC-30 ; antisense 50 -CAC ACT GAA GAA AAA AGG GGT TGC-30 ) designed from published gene sequences using Macvector 6.5.3 software (Oxford Molecular Group) and customermanufactured IDT (Integrated Technologies). The PCR reactions were performed in a 50 ml reaction volume, with 2 ml cDNA, 1 ml of sense and anti-sense primers (50 pmol/ml), 5 ml 10 buffer without MgCl2 (Invitrogen), 2 ml MgCl2, 1 ml 10 mM dNTP (Invitrogen), 0.5 ml Tag DNA polymerase (Invitrogen) and 37.5 ml DEPC water. The PCR amplification was carried out for 25 and 30 cycles, with the following steps: denature (94 C, 1 min), anneal (P4Hs: 55.0 C, 1 min) and extension (72 C, 2 min), using thermocycler (PE Biosystems). PCR products were separated by electrophoresis on 1% agarose gel (Biowhittaker), containing 50 ng/ml ethidium bromide (Sigma).
Miscellaneous Protein and RNA concentrations were determined by using ‘SOFTmaxPro’ analysis software and SPECTRA MAX Plus (Molecular Devices Co., Sunnyvale, CA, USA). The relative protein and mRNA levels were assessed by analysing band density using ‘Quantity One’ image analysis software (Biorad, Hercules, CA, USA) and normalization to GAPDH and b-actin expression allowed a semiquantitative comparison between samples.
Statistics Statistical analysis was performed using the Student’s t-test and one-way ANOVA; a probability level of <0.05 was required for statistical significance.
P4Hs is up-regulated in an acute bladder obstruction
Results Two-dimensional gel electrophoresis of human bladder smooth muscle cells To evaluate proteins that regulate the production of extracellular matrix under normal and pathologic conditions of the bladder, 2-DE was performed with the HBSMC. We identified, by mass spectrometry, 437 proteins from 476 spots (91.8%) that were differentially expressed in normal and highly pressurized (i.e. hydrostatic pressure of 40 cm for 3 days) HBSMC. Among them, 48 unique proteins significantly increased or decreased under high-pressure conditions when compared to controls. Among these individual protein spots of HBSMC, a prominent spot for the production of extracellular matrix was differentially expressed under normal and pathologic conditions; this spot highly correlated with P4Hs (data not shown).
Protein expression of prolyl 4 hydroxylase in vitro and in vivo As shown in Fig. 1, exposure of HBSMC and HBF to a sustained hydrostatic pressure resulted in the increased expression of P4Hs in a time- and pressure-dependent manner. In contrast to HBF, HBSMC demonstrated no significant difference in P4Hs protein expression at a hydrostatic pressure of 20 cm relative to duration of pressure, but at a hydrostatic pressure of 40 cm P4Hs protein expression significantly increased with the duration of pressure in both HBSMC and HBF. Both HBSMC and HBF demonstrated significantly higher P4Hs protein expression at a hydrostatic pressure of 40 cm compared to the lower pressure of 20 cm. To determine whether the results from acute obstructed bladder tissues in vivo would correlate with the results of the in-vitro study, Western blot analysis was carried out using control and acutely obstructed porcine bladder tissues. A significant up-regulation of P4Hs expression compared to controls was noted after exposure to a sustained hydrostatic pressure of 20 cm (Fig. 2).
Expression of mRNA prolyl 4 hydroxylase in vitro and in vivo To determine the effects of sustained pressure on P4Hs gene expression, we performed RT-PCR for P4Hs. As shown in Fig. 3, in vitro exposure of both HBSMC and HBF to a sustained hydrostatic pressure resulted in increased expression of P4Hs in a timeand pressure-dependent manner. With regard to
229 duration of pressure, P4Hs mRNA expression for both HBSMC and HBF was noted to demonstrate a significant difference at hydrostatic pressures of both 20 and 40 cm (P < 0.05) (Fig. 3). Additionally, P4Hs mRNA expression of HBF and HBSMC at a hydrostatic pressure of 40 cm demonstrated a significant increase compared to that at 20 cm (P < 0.05) (Fig. 3). Subsequently, to determine whether results from acute obstructed bladder tissues in vivo correlated with the results of the in vitro study, RT-PCR analysis was carried out using control and acute obstructed porcine bladder tissues. A significant up-regulation of P4Hs was noted after exposure to a sustained hydrostatic pressure of 20 cm; P4Hs showed a 22.4% increase in expression under these conditions relative to controls (P < 0.0001) (Fig. 4).
Discussion High intravesical pressure, originating from either intravesical obstruction or meningomyelocele, can lead to structural and functional changes of the urinary tract, and may lead to progressive damage to the bladder itself. From a biomechanical standpoint, it has been proposed that decreased compliance and capacity, due to pressure-induced bladder wall thickening, can result from an increase in the smooth muscle cell component and excess matrix deposition [9]. To achieve a better understanding of how increased intravesical pressure affects ultimate bladder compliance, it is important to determine not only the direct effects on transcription of the extracellular matrix components but also evaluate the molecular mechanisms responsible for the turnover of the extracellular matrix. For this reason, as a preliminary study, we applied 40 cm of hydrostatic pressure to HBSMC to evaluate possible pathological alterations that may affect extracellular matrix composition of the bladder wall. 2-DE was performed in HBSMC to quantify the proteins regulating the production of extracellular matrix under normal and pathologic conditions. Our results show that, among several individual protein spots, a prominent spot for the production of extracellular matrix highly correlated with P4Hs. P4Hs, an enzyme localized to the endoplasmic reticulum, plays a critical role in the synthesis of the extracellular matrix, and catalyzes the co-and posttranslational hydroxylation of proline residues in Xaa-Pro-Gly sequences of collagen, and other proteins with collagen-like sequences [10e12].
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The active enzyme is a tetramer (a2b2) consisting of three types of monomer with molecular weights of about 64,000 (a-subunit) and 60,000 (b-subunit). Three isoforms of the catalytic a-subunit, a (I), 2
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Figure 2 Western blot analysis of P4Hs in porcine bladder tissue. Lanes 1e5, controls; lanes 6e11, acute obstructed group.
a (II) and a (III), have been cloned and characterized from human and mouse tissues, and have been shown to form [a (I)]2b2, [a (II)]2b2 and [a (III)]2b2 tetramers, called type I, II and III P4Hs, respectively [13,14]. In non-urologic evaluations, many studies have demonstrated a good correlation between the level of P4Hs activity and the rate of collagen synthesis in cultured cells, as well as in vivo under many physiological and pathological conditions [15e17]. To date urologic studies of alterations of this molecular key determinant of extracellular
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matrix synthesis have not been performed. For this reason, we studied the alterations of P4Hs in vitro (i.e. HBSMC and HBF) under the influence of variable hydrostatic pressure using a novel pressure device that controls for hydrostatic pressure. Acute obstructed porcine bladder tissues were subsequently evaluated to see if these changes could also be seen in vivo. Western blot analysis, from the in vitro study, showed that exposure of HBSMC and HBF to
a sustained hydrostatic pressure generally resulted in increased expression of P4Hs in a time- and pressure-dependent manner. Subsequent Western blot analysis of the acute obstructed bladder tissues in vivo showed a significant up-regulation of P4Hs after exposure to sustained hydrostatic pressure when compared to controls. The RT-PCR analysis demonstrated a significant up-regulation of P4Hs in both HBSMC and HBF following exposure to 20 and 40 cmH2O for 72 h.
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Subsequent RT-PCR analysis of the control and acutely obstructed porcine bladder tissues also demonstrated a significant up-regulation of P4Hs after exposure to a sustained hydrostatic pressure of 20 cm compared to controls. These findings were consistent with those of the Western blot analysis. Therefore, the findings from this study conclusively demonstrate that BOO causes transcriptional change at the mRNA level of P4Hs; this results in production of extracellular matrix including collagen, and the resultant up-regulation of P4Hs proteins favours accumulation of extracellular matrix. Previous data [6,18] support the concept that pressures of less than 40 cmH2O may result in profound molecular and cellular alterations that may lead to further bladder deterioration. Our data also support the concept that the exposure of HBSMC and HBF to increased hydrostatic pressure ranging from 20 to 40 cmH2O may have a negative effect on extracellular matrix composition, and the resultant compliance of the bladder wall. Further experiments evaluating P4Hs inhibition of bladder function will be of use in the prevention of increased extracellular matrix composition and resultant bladder deterioration.
Acknowledgements Supported by grants from the Showwalter foundation, Robert Garrett professorship and Riley Memorial Foundation, Indiana University. C.A. supported by The Scientific and Technical Research Council of Turkey.
References [1] Hanash S. Disease proteomics. Nature 2003;422:226. [2] Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000;21:1037. [3] Cardinale GJ, Udenfriend S. Prolyl hydroxylase. Adv Enzymol Relat Areas Mol Biol 1974;41:245. [4] Kivirikko KK, Risteli L. Biosynthesis of collagen and its alterations in pathological states. Med Biol 1976;54:159. [5] Pilajaniemi T, Myllya R, Kivirikko K. Prolyl 4-hydroxylase and its role in collagen synthesis. J Hepatol 1991;13:S2. [6] Backhaus BO, Kaefer M, Haberstroh KM, Hile K, Nagatomi J, Rink RC, et al. Alterations in the molecular determinants of bladder compliance at hydrostatic pressures less than 40 cm H2O. J Urol 2002;168:2600. [7] Cottrell JS. Protein identification by peptide mass fingerprinting. Pept Res 1994;7:115. [8] Witzmann FA, Baur MD, Fieno AM, Grant RA, Keough TW, Kornguth SE, et al. Proteomic analysis of simulated occupational jet fuel exposure in the lung. Electrophoresis 1999;20:3659. [9] Peters CA, Vasavada S, Dator D, Carr M, Shapiro E, Lapor H. The effect of obstruction on the developing bladder. J Urol 1992;148:491. [10] Koivu J, Myllyla R, Helaakoski T, Pihlajaniemi T, Tasanen K, Kivirikko KI. A single polypeptide acts both as the b subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase. J Biol Chem 1987;262:6447. [11] Annunen P, Helaakoski T, Myllyharju J, Veijola J, Pihlajaniemi T, Kivirikko KI. Cloning of the human prolyl 4-hydroxylase a subunit isoform a (II) and characterization of the type II enzyme tetramer. J Biol Chem 1997;272: 17342. [12] Winter AD, Page AP. Prolyl 4-hydroxylase is an essential procollagen-modifying enzyme required for exoskeleton formation and the maintenance of body shape in the nematode Caenorhabditis elegans. Mol Cell Biol 2000;20:4084. [13] Pekkala M, Hieta R, Kursula P, Kivirikko KI, Wierenga RK, Myllyharju J. Crystallization of the proline-rich-peptide binding domain of human type I collagen prolyl 4-hydroxylase. Acta Crystallogr D Biol Crystallogr 2003;59:940. [14] Kukkola L, Hieta R, Kivirikko KI, Myllyharjhu J. Identification and characterization of a third human, rat, and mouse collagen prolyl 4-hydroxylase isoenzyme. J Biol Chem 2003;278:47685. [15] Mio K, Yamashita M, Odake Y, Tamai H, Takada K. Coenzyme A stimulates collagen production in cultured fibroblasts; possible mechanisms in enzymatic and gene expression. Arch Dermatol Res 2001;293:522. [16] Takahashi Y, Takahashi S, Shiga Y, Yoshimi T, Miura T. Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells. J Biol Chem 2000;275:14139. [17] Abergel RP, Pizzurro D, Meeker CA, Lask G, Matsuoka LY, Minor RR, et al. Biochemical composition of the connective tissue in keloids and analysis of collagen metabolism in keloid fibroblast cultures. J Invest Dermatol 1985;84:384. [18] Haberstroh KM, Kaefer M, Retik AB, Freeman MR, Bizios R. The effects of sustained hydrostatic pressure on select bladder smooth muscle cell functions. J Urol 1999;162:2114.
Collagen prolyl 4-hydroxylase is up-regulated in an acute bladder outlet obstruction Sang Don Lee b, Cem Akbal a, Rosalia Miseeri a, Chaeyong Jung a, Richard Rink a, Martin Kaefer a,* a
Department of Urology, Riley Hospital for Children, Indiana University, 702 Barnhill Drive, Indianapolis, IN 46202, USA b College of Medicine, Pusan National University, Busan, South Korea Received 26 September 2005; accepted 21 March 2006 Available online 7 July 2006
KEYWORDS Prolyl 4-hydroxylase; Bladder smooth muscle; Bladder outlet obstruction
Abstract Objective: Compliance is primarily related to extracellular matrix deposition, and prolyl 4-hydroxylase (P4Hs) plays a critical role in the synthesis of the matrix. To study the alteration of P4Hs, under the influence of variable hydrostatic pressure, a novel pressure device was used to expose human bladder smooth muscle cells (HBSMC) and fibroblasts (HBF) to pressures in the physiologic range. We then studied acute obstructed porcine bladder tissues to see if changes can also be seen after in-vitro obstruction. Materials and methods: HBSMC and HBF were exposed to pressures at 0, 20 and 40 cmH2O for up to 72 h. In-vivo studies were carried out next, using six normal (control) and five obstructed porcine bladders. Pigs were exposed to a consistent hydrostatic pressure of 20 cm for 24 h after ligation of the urethra. We used 2-DE to compare protein profiling of HBSMC under normal and increased pressures. Other analyses were used to detect molecular alteration and altered expression of mRNA for P4Hs. Results: We identified 437 proteins from 476 spots (91.8%) obtained from HBSMC that were differentially expressed under normal and increased pressures. Under increased pressure, 48 unique proteins were significantly increased or decreased, and a prominent protein regulating extracellular matrix synthesis highly correlated with P4Hs. The exposure of both HBSMC and HBF to a sustained hydrostatic pressure resulted in the increased expression of P4Hs in a time- and pressure-dependent manner. In vivo, P4Hs expression was also significantly increased in the obstructed group. Conclusions: P4Hs is up-regulated, in the human bladder, time and pressure dependently. The alteration of P4Hs over a short period may significantly influence the synthesis of extracellular matrix in vivo and lead to decreased compliance.
* Correspondence author. Tel.: þ1 317 274 8896; fax: þ1 317 274 7481. E-mail address: [email protected] (M. Kaefer). 1477-5131/$30 ª 2006 Published by Elsevier Ltd on behalf of Journal of Pediatric Urology Company. doi:10.1016/j.jpurol.2006.03.011
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S.D. Lee et al. Our results also support the concept that bladder outlet obstruction, with resultant pressures of 40 cmH 2 O or less, results in molecular changes consistent with decreased compliance. ª 2006 Published by Elsevier Ltd on behalf of Journal of Pediatric Urology Company.
Introduction Bladder outlet obstruction (BOO) with intravesical pressures exceeding 40 cmH2O results in alterations of bladder physiology, including wall thickening, reduced compliance and decreased capacity. From a biomechanical standpoint, compliance is primarily related to extracellular matrix deposition. Information, at the level of the proteome, is necessary to unravel the critical changes in extracellular matrix deposition involved in the pathogenesis of BOO. Comparative studies of protein expression, under normal and pathologic conditions, have led to the identification of aberrantly expressed proteins that may represent important new markers [1]. Two-dimensional gel electrophoresis (2-DE) is a powerful research technique that makes possible simultaneous examination of hundreds of polypeptides in a tissue or cell sample [2]. To quantify the proteins regulating the production of extracellular matrix under normal and pathologic conditions, 2-DE was performed in human bladder smooth muscle cells (HBSMC). Among several individual HBSMC protein spots, a prominent spot for the production of extracellular matrix highly correlated with prolyl 4-hydroxylase (P4Hs). BOO causes overproduction of extracellular matrix containing collagen. Synthesis of collagen, the major component of the extracellular matrix, begins with the translocation of collagen messenger RNA (mRNA) on the ribosomes of the rough endoplasmic reticulum, and hydroxylation of its proline moiety to hydroxyproline by P4Hs. Proline hydroxylation is thought to stabilize the triple helix, Xaa-Pro-Gly, in the collagen molecule, and P4Hs is believed to be a key enzyme in the regulation of collagen synthesis [3e5]. To date there have been no reports on whether a mechanical stimulus, such as hydrostatic pressure, has an effect on P4Hs. Furthermore, the threshold pressure, above which alterations in molecular key enzymes for bladder compliance occur, has not been determined. To study the alteration of P4Hs, under the influence of variable hydrostatic pressures, we employed a novel pressure device [6] to expose HBSMC and human bladder fibroblasts (HBF) to
pressures in the physiologic range. Acute obstructed porcine bladder tissues were subsequently evaluated to see if these changes were also seen as a result of in vitro obstruction.
Materials and methods Human bladder smooth muscle cells (HBSMC) and fibroblasts Human bladder smooth muscle cells (HBSMC) (Clonetics, East Rutherford, NJ, USA) and Human bladder fibroblast (HBF) were expanded in culture using smooth muscle cell basal media (SmBM, Clonetics) containing fetal bovine serum (5%), insulin (5 mg/ml), human recombinant fibroblast growth factor-b (2 ng/ml), human recombinant epidermal growth factor (0.5 ng/ml), gentamycin (15 mg/ml) and amphotericin (7.5 ng/ml) according to the manufacturer’s protocol. These cells were plated on P100 culture dishes and cultured for 2 days under standard cell-culture conditions (a sterile, humidified, 37 C, 95% air/5% CO2 environment). Thereafter, the media was changed to serum-free Leibovitz media (Invitrogen) and cells were placed in a pressure chamber under pressures of 20 and 40 cmH2O for 24, 48 and 72 h.
Computer-operated pressure system To expose the cultured HBSMC and HBF to a controlled stimulus of increased hydrostatic pressure, we used a custom-made, computer-operated pressure system, previously described by Backhaus et al. [6]. Briefly, a computer with software specially developed for this system controlled and maintained a designated pressure environment inside a sealed, acrylic chamber that housed standard tissue-culture plates of HBSMC and HBF. A computer interface controlled pressure levels, maintained (with feedback adjustments) the desired pressure for the duration of the experiments and collected pressure data. The prescribed pressure inside the chamber was monitored and maintained using a pressure transducer, and inlet and outlet solenoid valves that controlled the flow of compressed air mixture into and out of the system.
P4Hs is up-regulated in an acute bladder obstruction During experiments with HBSMC and HBF, the pressure chamber was placed in an incubator without involvement of CO2, and cells were kept in Leibovitz’s media designed for growth of cells in a CO2-free environment. This set up eliminates effects of pH change.
Experimental animals Following approval from the Institutional Animal Care and Use Committee (IACUC), the in vivo study was carried out using male pigs weighing an average of 29.8 kg. The animals were maintained in pathogen-free animal facilities according to IACUC guidelines.
227 the tissues, protein fractions were prepared by grinding the frozen tissue samples in a mortar and pestle. The pulverized tissues were homogenized with Poly Tron PT-MR2100 (Kinematica AG, Littau-Luzem, Switzerland) in modified RIPA buffer. The homogenates were centrifuged for 10 min at 14,000 g, 4 C. The pellet was resuspended and re-homogenized in modified RIPA buffer with 5% Triton X, and then incubated for 1 h on ice. The final homogenates were centrifuged for 2 min at 14,000 g, 4 C, and the supernatant (solubilized protein) was isolated and stored at 80 C until needed.
Two-dimensional gel electrophoresis and protein quantification
Surgical procedure The pigs were anesthetized using inhalation halothane, and a left paramedian incision was made in the lower abdomen. The urethra was exposed, and a No.1 silk suture was tied around the urethra. A new bladder outlet was created by the placement of a supra-pubic catheter into the bladder; into the lumen of this catheter a valve was placed that allowed urine to flow out of the bladder when a pre-determined pressure differential was generated. Valves with opening resistances of 20 cmH2O were used. Implantable, radiotelemetered manometry devices were placed into the bladder and abdomen to provide realtime ambulatory urodynamic assessment. The incision was closed with surgical sutures, and the pigs were housed in separate cages for 24 h. After 24 h of bladder outlet obstruction the pigs were killed. The body of the urinary bladder (tissue above the ureteral orifice) was dissected free of the serosa and surrounding fat. The bladder was divided in the midsagittal plane, and then cut into longitudinal smooth muscle strips (w5 20 mm). The muscle strips were rapidly frozen and stored at 80 C until needed.
Preparation of protein extracts Following exposure of cultured cells (HBSMC and HBF) to the hydrostatic pressure stimulus, the culture medium was removed, and cells were washed with 10 ml of ice-cold phosphate-buffered saline (PBS, pH 7.4) and lysed in modified RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, plus proteinase inhibitors). The cells were then scraped off the plates, transferred to a centrifuge tube, and centrifuged at 14,000 g, 4 C for 10 min. For
Sample preparation Harvested HBSMC were disrupted with a cocktail of 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris (hydroxymethyl) aminomethane, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The resulting cell lysates were subjected to physical shearing by passing through a syringe fitted with a 21-G needle, followed by syringes with 25-G and 27-G needles successively, and the addition of 50 mg/mL DNase I and 50 mg/mL RNase A. The sample was then centrifuged using a Beckman TL-100 Tabletop Ultracentrifuge (Palo Alto, CA) at 85,000 rpm for 2 h at 15 C. Two-dimensional gel electrophoresis The first dimension isoelectric focusing (IEF) was performed on precast 18-cm immobilized pH gradient (IPG) strips (Amersham Pharmacia Biotech) at 15 C with a maximum current setting of 50 mA/strip using an Amersham Pharmacia IPGphor IEF unit. The strips were rehydrated for a minimum of 10 h in ceramic strip holders in 350 mL of sample containing 7 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF, 20 mM dithiothreitol (DTT), and 0.5% IPG buffer (Amersham Pharmacia Biotech). The amount of protein loaded was w150 g for analytical gels and w350 mg protein for preparative gels. A low voltage of 30 V was applied during rehydration. After rehydration, the IEF run was carried out under the following conditions: 500 V/500 V h, 1000 V/1000 V h, and 8000 V/32000 V h. Voltage increases were performed on a step-wise basis. Before carrying out the second-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the strips were subjected to a two-step equilibration. The first was with an equilibration buffer
228 consisting of 6 M urea, 30% glycerol, 2% SDS, 50 mM TriseHCl (pH 6.8), 1% w/v DTT. The second step was with a buffer consisting of 6 M urea, 30% glycerol, 2% SDS, 50 mM TriseHCl (pH 8.8), 2.5% w/v iodoacetamide (Sigma). After the IPG strips were transferred onto the second-dimensional SDS-PAGE gel, the strips were sealed in place with 0.75% agarose. SDS-PAGE was performed on 1.0-mm thick 10% polyacrylamide gels at a constant voltage of 110 V at 10 C using an Amersham Pharmacia Iso-Dalt electrophoresis unit. Protein digestion and MALDI-MS analysis Protein identification by peptide mass fingerprinting [7] was carried out as described in detail previously [8]. Briefly, altered proteins were excised from replicate gels and digested with trypsin. The masses of the resulting peptides were determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Database searching and identification of proteins The proteins were identified by searching in SWISSPROT and NCBI non-redundant databases using MS-Fit (Protein prospector, UCSF, San Francisco, CA, USA). All mass searches were performed using a mass window between 1000 and 100,000 Da. The search parameters allowed for oxidation of methionine, N-terminal acetylation, carboxyamidomethylation of cysteins, and phosphorylation of serine, threonine and tyrosine. The criteria for positive identification of proteins were set as follows: at least four matching peptide masses and 50 ppm or better mass accuracy, and molecular weight and isoelectric point (pI) of identified proteins were required to match estimated values obtained from the image analysis.
Western blot analysis To determine P4Hs expression, the membrane proteins extracted from whole-cell lysates and bladder tissues were loaded onto 4e12% Novex Trise glycine electrophoresis gels (Invitrogen) for separation at 125 V, before blotting onto a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Membranes were blocked with 5% nonfat milk power in PBS at 25 C for 1 h before incubation for 2 h at 25 C with mouse antihuman-prolyl 4 hydroxylase (b-subunit) monoclonal antibody (Chemicon) at a dilution of 1:1000. After washing the membrane, a further incubation for 1 h at 25 C with anti-mouse horseradish
S.D. Lee et al. peroxidase-conjugated secondary antibody (Pierce) was performed before visualization using chemiluminescence substrate (Pierce).
Reverse transcription-polymerase chain reaction (RT-PCR) To determine the expression of mRNA for P4Hs, total RNA was extracted from porcine bladder tissues, HBSMC and HBF using TRIZOL solution (Invitrogen) according to the manufacturer’s protocol. RT was performed in a total reaction volume of 20 ml using 1 mg of total RNA, 1 ml Supercript II (Moloney’s murine leukemia virus reverse transcriptase, Invitrogen) and 100 pmol of oligo (dT)15 as a first-strand primer at 42 C for 1 h. PCR was performed using primers (sense 50 -GAA ACG GAT AAT ACG AGG CTT GTC-30 ; antisense 50 -CAC ACT GAA GAA AAA AGG GGT TGC-30 ) designed from published gene sequences using Macvector 6.5.3 software (Oxford Molecular Group) and customermanufactured IDT (Integrated Technologies). The PCR reactions were performed in a 50 ml reaction volume, with 2 ml cDNA, 1 ml of sense and anti-sense primers (50 pmol/ml), 5 ml 10 buffer without MgCl2 (Invitrogen), 2 ml MgCl2, 1 ml 10 mM dNTP (Invitrogen), 0.5 ml Tag DNA polymerase (Invitrogen) and 37.5 ml DEPC water. The PCR amplification was carried out for 25 and 30 cycles, with the following steps: denature (94 C, 1 min), anneal (P4Hs: 55.0 C, 1 min) and extension (72 C, 2 min), using thermocycler (PE Biosystems). PCR products were separated by electrophoresis on 1% agarose gel (Biowhittaker), containing 50 ng/ml ethidium bromide (Sigma).
Miscellaneous Protein and RNA concentrations were determined by using ‘SOFTmaxPro’ analysis software and SPECTRA MAX Plus (Molecular Devices Co., Sunnyvale, CA, USA). The relative protein and mRNA levels were assessed by analysing band density using ‘Quantity One’ image analysis software (Biorad, Hercules, CA, USA) and normalization to GAPDH and b-actin expression allowed a semiquantitative comparison between samples.
Statistics Statistical analysis was performed using the Student’s t-test and one-way ANOVA; a probability level of <0.05 was required for statistical significance.
P4Hs is up-regulated in an acute bladder obstruction
Results Two-dimensional gel electrophoresis of human bladder smooth muscle cells To evaluate proteins that regulate the production of extracellular matrix under normal and pathologic conditions of the bladder, 2-DE was performed with the HBSMC. We identified, by mass spectrometry, 437 proteins from 476 spots (91.8%) that were differentially expressed in normal and highly pressurized (i.e. hydrostatic pressure of 40 cm for 3 days) HBSMC. Among them, 48 unique proteins significantly increased or decreased under high-pressure conditions when compared to controls. Among these individual protein spots of HBSMC, a prominent spot for the production of extracellular matrix was differentially expressed under normal and pathologic conditions; this spot highly correlated with P4Hs (data not shown).
Protein expression of prolyl 4 hydroxylase in vitro and in vivo As shown in Fig. 1, exposure of HBSMC and HBF to a sustained hydrostatic pressure resulted in the increased expression of P4Hs in a time- and pressure-dependent manner. In contrast to HBF, HBSMC demonstrated no significant difference in P4Hs protein expression at a hydrostatic pressure of 20 cm relative to duration of pressure, but at a hydrostatic pressure of 40 cm P4Hs protein expression significantly increased with the duration of pressure in both HBSMC and HBF. Both HBSMC and HBF demonstrated significantly higher P4Hs protein expression at a hydrostatic pressure of 40 cm compared to the lower pressure of 20 cm. To determine whether the results from acute obstructed bladder tissues in vivo would correlate with the results of the in-vitro study, Western blot analysis was carried out using control and acutely obstructed porcine bladder tissues. A significant up-regulation of P4Hs expression compared to controls was noted after exposure to a sustained hydrostatic pressure of 20 cm (Fig. 2).
Expression of mRNA prolyl 4 hydroxylase in vitro and in vivo To determine the effects of sustained pressure on P4Hs gene expression, we performed RT-PCR for P4Hs. As shown in Fig. 3, in vitro exposure of both HBSMC and HBF to a sustained hydrostatic pressure resulted in increased expression of P4Hs in a timeand pressure-dependent manner. With regard to
229 duration of pressure, P4Hs mRNA expression for both HBSMC and HBF was noted to demonstrate a significant difference at hydrostatic pressures of both 20 and 40 cm (P < 0.05) (Fig. 3). Additionally, P4Hs mRNA expression of HBF and HBSMC at a hydrostatic pressure of 40 cm demonstrated a significant increase compared to that at 20 cm (P < 0.05) (Fig. 3). Subsequently, to determine whether results from acute obstructed bladder tissues in vivo correlated with the results of the in vitro study, RT-PCR analysis was carried out using control and acute obstructed porcine bladder tissues. A significant up-regulation of P4Hs was noted after exposure to a sustained hydrostatic pressure of 20 cm; P4Hs showed a 22.4% increase in expression under these conditions relative to controls (P < 0.0001) (Fig. 4).
Discussion High intravesical pressure, originating from either intravesical obstruction or meningomyelocele, can lead to structural and functional changes of the urinary tract, and may lead to progressive damage to the bladder itself. From a biomechanical standpoint, it has been proposed that decreased compliance and capacity, due to pressure-induced bladder wall thickening, can result from an increase in the smooth muscle cell component and excess matrix deposition [9]. To achieve a better understanding of how increased intravesical pressure affects ultimate bladder compliance, it is important to determine not only the direct effects on transcription of the extracellular matrix components but also evaluate the molecular mechanisms responsible for the turnover of the extracellular matrix. For this reason, as a preliminary study, we applied 40 cm of hydrostatic pressure to HBSMC to evaluate possible pathological alterations that may affect extracellular matrix composition of the bladder wall. 2-DE was performed in HBSMC to quantify the proteins regulating the production of extracellular matrix under normal and pathologic conditions. Our results show that, among several individual protein spots, a prominent spot for the production of extracellular matrix highly correlated with P4Hs. P4Hs, an enzyme localized to the endoplasmic reticulum, plays a critical role in the synthesis of the extracellular matrix, and catalyzes the co-and posttranslational hydroxylation of proline residues in Xaa-Pro-Gly sequences of collagen, and other proteins with collagen-like sequences [10e12].
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a (II) and a (III), have been cloned and characterized from human and mouse tissues, and have been shown to form [a (I)]2b2, [a (II)]2b2 and [a (III)]2b2 tetramers, called type I, II and III P4Hs, respectively [13,14]. In non-urologic evaluations, many studies have demonstrated a good correlation between the level of P4Hs activity and the rate of collagen synthesis in cultured cells, as well as in vivo under many physiological and pathological conditions [15e17]. To date urologic studies of alterations of this molecular key determinant of extracellular
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RT-PCR of P4Hs in HBF (A) and HBSMC (B).
matrix synthesis have not been performed. For this reason, we studied the alterations of P4Hs in vitro (i.e. HBSMC and HBF) under the influence of variable hydrostatic pressure using a novel pressure device that controls for hydrostatic pressure. Acute obstructed porcine bladder tissues were subsequently evaluated to see if these changes could also be seen in vivo. Western blot analysis, from the in vitro study, showed that exposure of HBSMC and HBF to
a sustained hydrostatic pressure generally resulted in increased expression of P4Hs in a time- and pressure-dependent manner. Subsequent Western blot analysis of the acute obstructed bladder tissues in vivo showed a significant up-regulation of P4Hs after exposure to sustained hydrostatic pressure when compared to controls. The RT-PCR analysis demonstrated a significant up-regulation of P4Hs in both HBSMC and HBF following exposure to 20 and 40 cmH2O for 72 h.
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S.D. Lee et al. 1 2 3 4 5 6
7 8 9 10 11 Prolyl 4-hydroxylase
Relative prolyl 4-hydroxylase/ β actin
ß-actin 1.4
*p<0.0001
1.3 1.2 1.1
n=5
1.0 0.9
n=6 Control
Figure 4
*: Student's
t-test
Acute obstructed bladder
RT-PCR of P4Hs in porcine bladder tissue.
Subsequent RT-PCR analysis of the control and acutely obstructed porcine bladder tissues also demonstrated a significant up-regulation of P4Hs after exposure to a sustained hydrostatic pressure of 20 cm compared to controls. These findings were consistent with those of the Western blot analysis. Therefore, the findings from this study conclusively demonstrate that BOO causes transcriptional change at the mRNA level of P4Hs; this results in production of extracellular matrix including collagen, and the resultant up-regulation of P4Hs proteins favours accumulation of extracellular matrix. Previous data [6,18] support the concept that pressures of less than 40 cmH2O may result in profound molecular and cellular alterations that may lead to further bladder deterioration. Our data also support the concept that the exposure of HBSMC and HBF to increased hydrostatic pressure ranging from 20 to 40 cmH2O may have a negative effect on extracellular matrix composition, and the resultant compliance of the bladder wall. Further experiments evaluating P4Hs inhibition of bladder function will be of use in the prevention of increased extracellular matrix composition and resultant bladder deterioration.
Acknowledgements Supported by grants from the Showwalter foundation, Robert Garrett professorship and Riley Memorial Foundation, Indiana University. C.A. supported by The Scientific and Technical Research Council of Turkey.
References [1] Hanash S. Disease proteomics. Nature 2003;422:226. [2] Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000;21:1037. [3] Cardinale GJ, Udenfriend S. Prolyl hydroxylase. Adv Enzymol Relat Areas Mol Biol 1974;41:245. [4] Kivirikko KK, Risteli L. Biosynthesis of collagen and its alterations in pathological states. Med Biol 1976;54:159. [5] Pilajaniemi T, Myllya R, Kivirikko K. Prolyl 4-hydroxylase and its role in collagen synthesis. J Hepatol 1991;13:S2. [6] Backhaus BO, Kaefer M, Haberstroh KM, Hile K, Nagatomi J, Rink RC, et al. Alterations in the molecular determinants of bladder compliance at hydrostatic pressures less than 40 cm H2O. J Urol 2002;168:2600. [7] Cottrell JS. Protein identification by peptide mass fingerprinting. Pept Res 1994;7:115. [8] Witzmann FA, Baur MD, Fieno AM, Grant RA, Keough TW, Kornguth SE, et al. Proteomic analysis of simulated occupational jet fuel exposure in the lung. Electrophoresis 1999;20:3659. [9] Peters CA, Vasavada S, Dator D, Carr M, Shapiro E, Lapor H. The effect of obstruction on the developing bladder. J Urol 1992;148:491. [10] Koivu J, Myllyla R, Helaakoski T, Pihlajaniemi T, Tasanen K, Kivirikko KI. A single polypeptide acts both as the b subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase. J Biol Chem 1987;262:6447. [11] Annunen P, Helaakoski T, Myllyharju J, Veijola J, Pihlajaniemi T, Kivirikko KI. Cloning of the human prolyl 4-hydroxylase a subunit isoform a (II) and characterization of the type II enzyme tetramer. J Biol Chem 1997;272: 17342. [12] Winter AD, Page AP. Prolyl 4-hydroxylase is an essential procollagen-modifying enzyme required for exoskeleton formation and the maintenance of body shape in the nematode Caenorhabditis elegans. Mol Cell Biol 2000;20:4084. [13] Pekkala M, Hieta R, Kursula P, Kivirikko KI, Wierenga RK, Myllyharju J. Crystallization of the proline-rich-peptide binding domain of human type I collagen prolyl 4-hydroxylase. Acta Crystallogr D Biol Crystallogr 2003;59:940. [14] Kukkola L, Hieta R, Kivirikko KI, Myllyharjhu J. Identification and characterization of a third human, rat, and mouse collagen prolyl 4-hydroxylase isoenzyme. J Biol Chem 2003;278:47685. [15] Mio K, Yamashita M, Odake Y, Tamai H, Takada K. Coenzyme A stimulates collagen production in cultured fibroblasts; possible mechanisms in enzymatic and gene expression. Arch Dermatol Res 2001;293:522. [16] Takahashi Y, Takahashi S, Shiga Y, Yoshimi T, Miura T. Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells. J Biol Chem 2000;275:14139. [17] Abergel RP, Pizzurro D, Meeker CA, Lask G, Matsuoka LY, Minor RR, et al. Biochemical composition of the connective tissue in keloids and analysis of collagen metabolism in keloid fibroblast cultures. J Invest Dermatol 1985;84:384. [18] Haberstroh KM, Kaefer M, Retik AB, Freeman MR, Bizios R. The effects of sustained hydrostatic pressure on select bladder smooth muscle cell functions. J Urol 1999;162:2114.