To determine the role of CypB in vivo, we targeted exon 3 by homologous recombination, and generated mice bearing the knockout allele (Figure 1A). The resulting allele contains an out of frame join between exons 2 and 4, thus was predicted to completely inactivate the gene. Mating of heterozygote mutant mice gave rise to viable homozygous knockouts at the expected Mendelian ratio. Successful deletion of the gene was verified by Southern blotting (Figure 1B) and by Western blotting (Figure 1C, Figure S1A). mRNA generated from the mutated allele accumulated to significantly lower levels than normal, most likely due to nonsense-mediated decay (Figure S1B).

Because CypB is expressed in all cell types, and is highly conserved from yeast to humans, we anticipated that homozygous loss might cause developmental abnormalities during embryogenesis. Surprisingly, CypB knockout mice appeared normal at birth, and both sexes were fertile. In addition, Ppib^−/− mothers had no apparent difficulties giving birth, feeding, or raising their pups. On the other hand, homozygous CypB knockout mice had reduced body size and weight, in comparison to littermate controls (Figure 1C and 1D), and typically died between 40 and 50 weeks of age of unclear etiology. A striking feature was pronounced kyphosis, noted as early as 8 weeks after birth that progressed in severity with age (Figure 1F, Figure 2A, Figure S2A). Dual-energy x-ray absorptiometry further demonstrated this kyphosis, and suggested that knockout mice had reduced bone density (data not shown). Although rhizomelia has been described in some types of osteogenesis imperfecta [4],[5], we did not observe differences in the ratio of femur to tibia lengths in CypB-deficient mice compared to littermate controls (0.819±0.027 vs. 0.826±0.013) (Figure S2B).

To study the pathophysiology of altered bone development, Ppib^−/− or littermate control mice were euthanized and femurs dissected for analysis by microcomputed tomography. Serial sections of femurs revealed dramatically reduced amounts of trabecular bone in mice lacking CypB (Figure 2B). Average bone volume was significantly reduced and the separation between trabeculae was increased in mutant mice (Figure 2C).

Because of the critical role for collagen in directing bone formation [21], we next analyzed collagen from Ppib^−/− mice, as was performed for CRTAP knockout mice [5]. Skin fibroblasts were prepared from knockout and littermate control embryos and maintained in culture. Collagen secreted into serum-free medium was collected and fractionated on SDS-PAGE. Western blotting revealed slightly delayed migration (Figure 3A), consistent with increased post-translational modification, as was noted previously in collagen from LEPRE1 or CRTAP mutant cells [3],[4].

Samples of bone and cartilage were extracted by limited pepsin digestion, and proteins resolved by SDS-PAGE. Coomassie blue stained bands were excised, subjected to trypsinization, and analyzed by tandem mass spectrometry. The tryptic peptide containing proline-986 (975-DGLNGLPGPIGPPGPR-990) was identified, and further analyzed by MS2 and MS3 for hydroxylation modifications (Figure 3B, Figure S3). Remarkably similar to collagen from LEPRE1 mutant humans and CRTAP knockout mice [5], multiple analyses demonstrated an almost complete absence of 3-hydroxy proline in the peptide at Pro-986 from CypB-knockouts, while collagen from wild type and heterozygote littermates had abundant amounts of peptide containing hydroxy-proline in this position. The known sites of prolyl–4 hydroxylation at residues 981 and 987 were appropriately modified by hydroxylation in the peptides from both wild type and knockout collagen. Similar results were obtained for Type II collagen from cartilage (Figure 3B).

3-hydroxylation of Pro-986 in type I collagen is required for correct fibril formation because mutation of P3H1 or CRTAP causes the accumulation of aberrant fibrils with wider than normal diameter [4],[5]. We therefore examined subcutaneous collagen fibrils in CypB knockout mice by transmission electron microscopy. The majority of collagen fibrils in these mice were on average 1.45 times wider than similar samples from littermate control mice (114.6 +/− 22.4 nm vs. 78.6 +/− 12.4 nm diameter) (Figure 3C). Because of the reported interaction between CypB and type I collagen precursors during their transit through the export pathway [19], we next stained cells for endogenous collagen. In the absence of ascorbic acid, the majority of collagen co-localized with the ER-resident marker PDI (Figure 4). Upon addition of ascorbic acid for 24 hours, collagen in wild type cells concentrated in the area of the golgi apparatus, as indicated by co-staining with a GM130 antibody [22]. However, in cells lacking CypB, a significant amount of collagen remained in the ER (Figure 4). Thus, CypB is important for appropriate subcellular localization of collagen within the protein secretion system.

Thin skin is a common feature of patients with OI. We noticed that CypB −/− mice were easily identified by their loose skin (Figure 5A). To determine whether this was related to a defect in collagen, sections of skin were fixed and stained by H & E, or Sirrius Red (Figure 5B and 5C). Cellular appearance was normal, however we noted decreased intensity of Sirrius red staining, suggesting lower concentrations of collagen in CypB-deficient skin. Total collagen was extracted from skin samples with pepsin digestion, and tested by ELISA for type I collagen content, verifying this result (Figure 5D). To quantitate the defect further, we performed tensile strength testing of skin samples, as was done previously for Tenascin-X deficient mice, which are a model for Ehlers−Danlos syndrome [23]. Comparison of stress versus strain (Figure 5E) revealed several abnormal properties of CypB−/− skin samples, which were very similar to published results from Tenascin-X knockout mice. The ‘toe’ region of the stress-strain curves, representing normal ranges of skin stretching, were much longer than normal (Figure 5F). The steep linear portion of the curves had flatter slopes (Figure 5G), demonstrating dramatically lower stiffness of the aligned collagen fibers, thought to be due to the density and cross-linking of the fibrils [23]. Lastly, much lower forces were required to break CypB-knockout mouse skin (Figure 5H). All three parameters were essentially the same between wild type and heterozygote mice.

Lastly, we prepared collagen from skin samples, and analyzed it by mass spectroscopy. As described for bone and cartilage collagen, there was a severe loss of triple-hydroxylated collagen representing the 986-hydroxylated peptide (Figure S4).

Several other investigators have reported that CypB is able to bind to collagen, and to the P3H1 / CRTAP complex [5],[8]. To verify this in our system, we prepared recombinant CypB as a GST-fusion protein from E.coli, and tested its ability to bind to collagen in the form of gelatin linked to sepharose. GST-CypB indeed bound stably to gelatin-sepharose, and there was no interaction between the control protein GST and gelatin (Figure 6A). Addition of cyclosporine A (CsA) completely blocked the CypB-gelatin interaction, indicating that the enzymatic active site of CypB (which binds to the twisted proline intermediate during the cis-trans peptidyl prolyl isomerase reaction [24]) is important for affinity to collagen.

We investigated whether recombinant CypB would also bind to P3H1 from cells. Lysates from wild type fibroblasts were prepared, incubated with the fusion protein, and then passed over glutathione-agarose resin to recover GST-CypB. We observed a moderate degree of retention of P3H1 specifically to GST-CypB, and none to GST alone, indicating they may indeed interact. (Figure 6B). However, we noted that Type I collagen also was retained specifically in these pull-down experiments, raising the alternative possibility that P3H1 might be indirectly associating with CypB through collagen monomers acting as intermediate bridges. To further explore this latter possibility, we repeated the pull-down experiments in the presence of CsA, to inhibit CypB binding to collagen. However, CsA had only a modest inhibitory effect on the CypB-P3H1 association (Figure 6C), suggesting that the proline-binding site of CypB is probably not involved in interaction with P3H1. Interestingly, GST-CypB pulled down a much larger fraction of cellular P3H1 when added to lysates from Ppib−/− cells, consistent with the idea that under normal conditions, most P3H1 is already bound to endogenous CypB (Figure 6C).

In conducting these studies, we also found that the amount of P3H1 was relatively reduced in CypB-deficient cells (Figure 6D). This effect was specific because there was not a similar reduction in other ER-resident proteins, such as PDI, FKBP65, HSP47. However, the residual P3H1 retained its ability to bind to collagen, as determined by affinity to gelatin-sepharose (Figure 6E). Thus, stable accumulation of P3H1 in fibroblasts depends upon CypB, however it does not require CypB in order to associate with collagen.

To investigate the reciprocal relationship of P3H1 effects on CypB, several commercial shRNA lentivirus preparations were tested, and two were found to cause significant depletion of P3H1 protein in murine fibroblasts. We found that in both cases, reduction of P3H1 did not have any effect on intracellular levels of CypB (Figure 7A). On the other hand, knockdown of P3H1 severely reduced the amount of CypB that could bind to gelatin-sepharose in vitro, indicating that a substantial portion of CypB-gelatin binding depends upon the P3H1 protein. This result was obtained regardless of whether the cells were grown in the presence or absence of ascorbic acid (Figure 7B), which is known to increase collagen production and hydroxylation [25],[26].

Like P3H1, CRTAP has been reported to be mutated in mice and humans with OI. CRTAP does not have a known enzymatic function, and, although collagen from CRTAP−/− mice was shown to have reduced prolyl–3-hydroxylation, its mechanism in this process is not understood. Unfortunately, we were unable to obtain antibodies that recognized the murine CRTAP, thus we turned to a human cell system to explore its potential interactions. HeLa cells were found to have easily detectable CRTAP. CypB was knocked down using a shRNA lentivirus, and lysates were tested as described above. Although CypB was efficiently reduced, there was no impairment of either CRTAP accumulation, nor of the binding of CRTAP to gelatin-sepharose (Figure 7C).

Knockdown of CRTAP in HeLa cells, on the other hand, caused a substantial depletion of P3H1 (Figure 7D). In addition, although there was not an effect of loss of CRTAP on CypB intracellular levels, we did observe a dramatic impairment in the ability of CypB from these lysates to bind to gelatin-sepharose (Figure 7D). We conclude that CRTAP maintains the appropriate cellular accumulation of P3H1, and facilitates CypB binding to collagen.

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee. A targeting plasmid was generated using a BAC clone containing the entire Ppib gene from 129SvJ genomic DNA. Germline transmission in 129 x C56B6 mice was achieved essentially as previously described [44]. The initial mice carried a conditional knockout allele (Figure 1) in case total deletion would be embryonic lethal. Mice were crossed to MMTV-Cre transgenic mice to cause deletion of the floxed exon 3 in female germ cells, after which the Cre transgene was bred out of the offspring, since it was no longer needed. Pups from these females were heterozygous for the KO allele, and were bred as needed to generate heterozygote or homozygote mutant, or littermate control wild type mice.

Mouse embryonic fibroblasts (MEFs) or skin fibroblasts were generated as previously described [45]. Cells were maintained in DMEM +10% fetal calf serum with 50 µg/ml ascorbate (sigma) unless otherwise indicated. For isolation of secreted collagen, cells were cultured in serum free DMEM containing ascorbate. For knockdown in skin fibroblasts, Lentiviral vectors encoding small hairpin interfering RNA sequences (shRNA) were obtained from sigma (LEPRE1 and CypB) or Open Biosystems (CRTAP). Lentiviral vectors were transfected in 293 FT cells with packaging plasmids (Invitrogen) for generating lentiviral particles. Skin fibroblasts were transduced with specific lentiviruses and selected by 2 ug/ml puromycin (Invitrogen).

Mice were euthanized and analyzed by radiography (GE AMX 4) of whole body or hind limbs. The femur was cleaned of soft tissue and measured ex-vivo on a uCT 35 micro-CT scanner (Scanco Medical, Basserdorf,Switzerland). Trabecular architecture was measured distally from the growth plate (70 kVP, increment 7 um) at a resolution of 7 um. The analysis region was represented by 100 slices.

To analyze modification of secreted procollagen, confluent fibroblasts were stimulated in serum-free conditions containing ascorbate during the 48 hrs. The harvested media were electrophoresed on 5% SDS polyacrylamide gels under reducing conditions and processed for westernblotting with type I collagen antibody (SouthernBioTech). To examine the overall architecture and quantify the collagen content in a given area of mouse skin tissue, sections of paraffin-embedded skin sample from abdomen were stained with H&E or Picrosirius red (Polysciences, Inc.). Type I collagen in the extract from skin samples was quantified with Mouse Type I Collagen Detection Kit (Chondrex, Inc.)

To determine prolyl 3-hydroxylation in collagen samples from skin, bone and cartilage, collagen was solubilized and extracted by a limited digestion with pepsin (100 µg/ml; Calbiochem) in 0.5N acetic acid for 48 hr. This extracts were resolved by SDS-PAGE. For collagen samples obtained from skin fibroblast culture, the harvested culture media were concentrated by Centricon (Millipore) and resolved by SDS-PAGE. The Coomassie Blue stained SDS-PAGE gel bands were prepared for mass spectrometry analysis using the following procedures. The gel bands were destained with 50 mM Tris, pH 8.1/50% acetonitrile until nearly clear. The bands were then reduced with 30 mM DTT/50 mM Tris, pH 8.1 at 55°C for 40 minutes and alkylated with 40 mM iodoacetamide at room temperature for 40 minutes in the dark. Proteins were digested in-situ with 30 ul (0.004 ug/ul) trypsin (Promega Corporation, Madison WI) in 20 mM Tris pH 8.1/.0002% Zwittergent 3–16, at 37°C overnight followed by peptide extraction with 60 ul of 2% trifluoroacetic acid, then 60 ul of acetonitrile. The pooled extracts were concentrated to less than 5 ul on a SpeedVac spinning concentrator (Savant Intruments, Holbrook NY) and then brought up in 0.15% formic acid/0.05% trifluoroacetic acid for protein identification by nano-flow liquid chromatography electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) using a ThermoFinnigan LTQ Orbitrap Hybrid Mass Spectrometer (ThermoElectron Bremen, Germany) coupled to an Eksigent nanoLC-2D HPLC system (Eksigent, Dublin, CA). The digest peptide mixture was loaded onto a 250nl OPTI-PAK trap (Optimize Technologies, Oregon City, OR) custom packed with Michrom Magic C8 solid phase (Michrom Bioresources, Auburn, CA). Chromatography was performed using 0.2% formic acid in both the A solvent (98%water/2%acetonitrile) and B solvent (80% acetonitrile/10% isopropanol/10% water), and a 5%B to 45%B gradient over 60 minutes at 400 nl/min through a Michrom packed tip capillary Magic C18 75 µm x 200 mm column. The initial LTQ Orbitrap mass spectrometer experiment was set to perform a FT full scan from 375–1600 m/z with resolution set at 60,000 (at 400 m/z), followed by linear ion trap MS/MS scans on the top five ions. Dynamic exclusion was set to 2 and selected ions were placed on an exclusion list for 40 seconds. The lock-mass option was enabled for the FT full scans using the ambient air polydimethylcyclosiloxane (PCM) ion of m/z = 445.120024 or a common phthalate ion m/z = 391.284286 for real time internal calibration.

The experiment to target the hydroxyproline sites of the peptide DGLNGLPGPIGPPGPR, relied on performing MS3 on the abundant y-ion from fragmentation N-terminal to a proline residue. The Orbitrap full scan used a list of masses for the [M+2H]2+ ions of this peptide without and with 1 to 5 hydroxyprolines to trigger the data dependant ion trap MS/MS scans. The ion trap MS/MS/MS scans were triggered on the most abundant ion of the MS/MS scan. If no masses from the list were detected in the Orbitrap full scan, then the most abundant ions triggered the MS/MS events.

The MS/MS raw data were converted to DTA files using extract_msn.exe from Bioworks 3.2 and correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot databases using Mascot™ 2 (Matrix Sciences London, UK). All searches were conducted with fixed modification of carbamidomethyl-cysteine and variable modifications allowing oxidation of methionines for methione sulphoxide, oxidation of prolines for hydroxyproline, and protein N-terminal acetylation. The search was restricted to full trypsin generated peptides allowing for 2 missed cleavages and was left open to all species. Peptide mass search tolerances were set to 10 ppm and fragment mass tolerances are set to±0.8 Daltons. All protein identifications were considered when Mascot individual peptide scores were above the 95% percentile for probability and rank number one of all the hits for the respective MS/MS spectra. The MS/MS/MS scans were analyzed manually.

Skin tissues taken from lower abdomen were processed for transmission electron microscopy as described [46]. Skin biopsy was fixed overnight in 0.1% glutaraldehyde and 4% formaldehyde in 0.1-M phosphate buffer at pH 7.2. After primary fixation, tissues were rinsed in 0.1-M phosphate buffer, followed by postfixation in phosphate-buffered 1% osmium tetroxide. After rinsing in three changes of distilled water, the tissue was stained en bloc with 2% uranyl acetate at 55°C. The tissues were rinsed in distilled water, dehydrated in progressive concentrations of ethanol and propylene oxide, and embedded in Spurr resin. The resin was polymerized at 65°C. Thin sections were mounted on copper grids for evaluation with a transmission electron microscope (JEOL JEM-1400; JEOL USA, Peabody, Massachusetts). To analyze fibril morphology and density, we selected 5 adjacent regions lying just beneath the epidermal basement membrane from each of 4 sex- and age- matched wild type and CypB knockout littermates.

MEFs were cultured on coverslides, fixed with 2.5% paraformaldehyde in PBS and permeabilized using 0.2% Triton X-100 in PBS for 2 min. Cells were blocked with PBS containing 5% goat serum and stained for intracellular collagen with anti-mouse collagen antibody (AB765P; Chemicon). Cellular localization was verified by costaining with anti-PDI (stressgen) or anti-GM130 antibody (BD). Samples were visualized using a Zeiss epifluorescence microscope.

Cells were lysed in 1% NP-40, 20 mM HEPES [pH 7.4], 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, and 45 µg/ml aprotinin. 50 µg of protein lysates were resolved by 8% SDS-PAGE and transferred to charged nylon membranes (Millipore). Western blots were probed for cyclophilin B with rabbit polyclonal antibody (Affinity BioReagents) or mouse monoclonal antibody (k2E2; Abcam), type I collagen (SouthernBioTech), P3H1 (Abnova), PDI (Stressgen), Hsp47 (Stressgen), FKBP65 (BD), GST (Sigma), CRTAP (Abnova) and beta-actin (Sigma). Reactive bands were visualized with a secondary antibody conjugated with HRP (Zymed) and chemiluminescence (Thermo scientific). For pulldowns, cell extracts were mixed with GST-cyclophilin B or GST and treated with GSH-agarose (GE healthcare), washed in lysis buffer and eluted by SDS sample buffer. For binding assays with gelatin-sepharose (GE healthcare), lysates containing approximately 1 mg of total protein were mixed with gelatin-sepharose for 3 h at 4°C with continuous rotation. The resulting precipitate was washed twice with lysis buffer, subjected to SDS-PAGE, and then blotted for the indicated protein.

Total RNA was purified with TRIZOL (Invitrogen) from splenocytes or MEFs and used as a template for synthesis of cDNA with SuperScript III First Strand RT-PCR kit (Invitrogen). With TaqMAN Gene Expression Assays (Applied Biosystems), Ppib and Actin mRNA levels were measured on a ABI 7900HT Thermocycler (Applied Biosystems). All q-PCR reactions were run in quadriplicate. Ppib level were normalized to constitutively expressed actin. The graph of fold change was generated by ΔΔCt values.

Skin from 38-weeks old female mice was biomechanically tested for tensile strength as described [23]. Stress was calculated by normalizing measured force with cross-sectional area. The strain at a stress equivalent to 0.05 (Newtons/mm) was used as the toe length. Slope was calculated with data in the linear portion (above 0.2 Newtons/mm). Highest force was normalized with cross-sectional area for failure force.