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Keywords:

  • PEDF;
  • OSTEOGENESIS IMPERFECTA;
  • BONE MINERALIZATION;
  • OSTEOBLAST

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Osteogenesis imperfecta type VI (OI type VI) has recently be linked to a mutation in the SERPINF1 gene, which encodes pigment epithelium-derived factor (PEDF), a ubiquitously expressed protein originally described for its neurotrophic and antiangiogenic properties. In this study, we characterized the skeletal phenotype of a mouse with targeted disruption of Pedf. In normal mouse bone, Pedf was localized to osteoblasts and osteocytes. Micro–computed tomography (µCT) and quantitative bone histomorphometry in femurs of mature Pedf null mutants revealed reduced trabecular bone volume and the accumulation of unmineralized bone matrix. Fourier transform infrared microscopy (FTIR) indicated an increased mineral:matrix ratio in mutant bones, which were more brittle than controls. In vitro, osteoblasts from Pedf null mice exhibited enhanced mineral deposition as assessed by Alizarin Red staining and an increased mineral:matrix determined by FTIR analysis of calcified nodules. The findings in this mouse model mimic the principal structural and biochemical features of bone observed in humans with OI type VI and consequently provide a useful model with which to further investigate the role of PEDF in this bone disorder.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Osteogenesis imperfecta (OI) comprises a heterogeneous group of connective tissue disorders characterized by bone fragility with susceptibility to fractures. Most cases of OI are caused by dominant mutations in COL1A1 or COL1A2, the genes encoding the two type I procollagen alpha chains, proα1 (I) and proα2 (I).[1] Mutations in these genes result in quantitative and/or qualitative defects in type I collagen production by osteoblasts.[2] In addition to dominant collagen I mutations, other cases of recessive OI are caused by mutations in proteins that are collagen I modifiers and chaperones, such as the multiple members of the collagen 3-hydroxylation complex.[3-7]

OI type VI is a moderate to severe, recessive form of the disease in which patients typically present with fractures during infancy[8] and malformed bones, and frequently have limited mobility by the time they reach adolescence. OI type VI is distinct from other types of OI in that the affected subjects display an osteomalacia-like phenotype characterized by thickened osteoid and delayed mineralization. A distinct “fish-scale” lamellar appearance of the bone has also been noted in bone biopsies.[8] In addition, bisphosphonates are generally less effective in treating type VI than other subtypes of OI.[9]

The molecular basis of OI type VI was recently identified as an inactivating mutation in the SERPINF1 gene, which encodes pigment epithelium-derived factor (PEDF).[10-13] SERPINF1 resides on chromosome 17p13.3 and encodes a 1.5-kb transcript that is translated into a 50-kDa secreted glycoprotein.[14] Originally isolated from retinal epithelium and characterized for its neurotropic and antiangiogenic properties,[15, 16] PEDF is ubiquitously expressed in both human and mouse[17] and has been implicated in cell cycle control,[18] fat metabolism,[19] and tumorigenicity.[20] PEDF has been reported to bind the receptor adipose triglyceride lipase (ATGL/desnutrin/iPLA)[21] in hepatocytes and adipocytes, but the protein also contains binding sites for collagen type I, hyaluronan, the laminin receptor, and heparin, as well as a nuclear localization sequence.[14, 22]

In this study we characterized the bone abnormalities in mice lacking Pedf as a first step in determining its function in the skeleton. Our results show that loss of Pedf in mice produce skeletal features which resemble the main defects seen in patients with OI type VI. The availability of this mouse model should enable further studies to more completely understand the function of PEDF in bone and in other tissues in which it is expressed.

Subjects and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Mice

PEDF−/− mice were created as described.23 For all Pedf−/− mouse experiments, wild-type (WT) controls were drawn from littermates resulting from Pedf+/− × Pedf+/− matings. All procedures involving mice were approved by the Institutional Animal Care and Use Committee of The Johns Hopkins University.

Immunohistochemistry

Immunohistochemistry of bone sections was performed using a streptavidin–biotin complex immunoperoxidase technique. Sections were deparaffinized, treated to inhibit endogenous peroxidase, and subjected to antigen retrieval as described.[23] All tissues were stained with hematoxylin and eosin (H&E). An experienced pathologist performed all histological evaluations.

Micro–computed tomography and histomorphometry

The three-dimensional microarchitecture of the intact right femurs was evaluated using a SkyScan1172 high-resolution Micro–computed tomography (µCT) and its NRecon reconstruction software (Bruker MicroCT, Kontich, Belgium) in accordance with the recommendations of the American Society for Bone and Mineral Research (ASBMR).[24] Quantitative histomorphometry was conducted at distal femoral sites as described[25]; serial sections to the stained sections were left unstained for fluorescent microscopy. All parameters comply with the recommendations of the Histomorphometry Nomenclature Committee of the ASBMR.[26, 27]

Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) was performed on mouse femurs as described,[28] with the mineral:matrix ratio being calculated as the ratio of the integrated area under the phosphate band (920–1180 cm−1) to that of the amide I band (1585–1720 cm−1). Means and standard deviations for the three to five images in trabecular and cortical bone were calculated and compared. For the mineral formed in cultures, the culture dishes were air-dried and the mineral scraped from the dish by combination with dry KBr. Pellets formed from these mixtures were analyzed as described for tissue samples.

Mechanical testing

The strength of the femoral midshaft was assessed by three-point bending using a low-force mechanical testing system (Bose Electroforce 3100) according to established methods.[29-31] Force-displacement data were used to determine structural properties (ultimate force, ultimate displacement, stiffness, and energy-to-failure).

Osteoblast culture

Primary osteoblasts were isolated from calvarial bones from 1-day-old to 3-day-old neonatal pups via collagenase type I digestion according to standard techniques. Cells were differentiated by supplementing the media with 10 mM β-glycerol phosphate and 50 μg/mL ascorbic acid. Media was changed every 2 to 3 days. Alkaline phosphatase and Alizarin Red staining were carried out according to standard techniques. RNA was isolated using Trizol and prepared for quantitative RT-PCR analysis. Primer sequences used for to assess the expression of Pedf, Ank, Npp1, AnxA2, AnxA5, AnxA6, Dmp1, Phex, and Bsp are available upon request.

Statistical analysis

All experiments were repeated at least three times unless otherwise stated and results are expressed as mean ± SEM. For comparisons between two groups of data, the Student's t test was used; p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Immunohistochemical analysis performed on decalcified bone sections showed positive Pedf immunoreactivity in the osteoblasts lining trabecular surfaces, as well as embedded osteocytes (Fig. 1A). Positive staining was also observed in some osteocytes in cortical bone (not shown). Additionally, Pedf-positive immunoreactivity was evident in perivascular cells and the prehypertrophic chondrocytes of the growth plate (data not shown). As shown in Fig. 1B, increased CD-31 immunoreactivity was detected in vascular structures from the Pedf−/− bones, consistent with previous studies demonstrating antiangiogenic properties of Pedf.[16]

image

Figure 1. PEDF expression and loss of function in vivo. (A) Immunohistochemical staining of Pedf in osteoblasts (arrows) in trabecular bone from a wild-type (WT) mouse. (B) CD31 staining in bone from WT (a) and Pedf−/− mice (b). (C, D) Quantitative µCT analysis of the distal femur in WT and Pedf−/− mice at 12 weeks of age (n = 4–5 mice): (C) representative images; (D) bone volume/tissue volume, BV/TV (%). (EI) Histomorphometric analysis of the distal femoral metaphysis in male WT and Pedf−/− mice at 6 weeks of age (n = 5–7 mice): (E) osteoblast number per bone perimeter, Ob.N/BPM (number per 100 mm) at 6 weeks of age; (F) osteoclast number per bone perimeter at 6 weeks of age; (G) bone formation rate per bone surface, BFR/BS, mm3/cm2/year; (H) osteoid thickness (µm); (I) osteoid maturation time (days). (J) FTIR measures of mineral:matrix ratios in cortical and trabecular bone. (KM) Three-point bending analysis of femurs in WT and PEDF−/− mice (n = 5–8 mice): (K) ultimate force (N); (L) ultimate displacement (mm); (M) energy to failure (mJ). All error bars represent SEM. *p < 0.05.

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µCT analysis at 12 weeks of age showed that Pedf−/− mice had significantly reduced trabecular bone volume fraction in association with reduced trabecular numbers relative to WT littermates (Fig. 1C, D). Bone histomorphometry performed at 6 weeks of age revealed no significant differences in the numbers of osteoblasts or osteoclasts in the mutant mice when compared to WT controls (Fig. 1E, F). Likewise, bone formation rate was similar in mutant and WT mice (Fig. 1G). However, Pedf−/− mice exhibited significant increases in osteoid thickness (Fig. 1H) and osteoid maturation time (Fig. 1I), suggestive of a defect in the mineralization process. Analysis of bone mineral properties using FTIR demonstrated that, like other models of OI,[28] the mineral:matrix ratio was significantly increased in Pedf−/− mice (Fig. 1J). The loss of Pedf function did not affect parameters of cortical bone structure (data not shown) but assessment of cortical bone strength in three-point bending experiments suggested that the bones of Pedf−/− mice were more brittle that those of WT littermates mice at 12 weeks of age. Ultimate displacement and energy to failure were significantly reduced in bones from mutant mice (Fig. 1KM), possibly as a result of the increased mineral:matrix ratio in this bone envelope.

To more directly analyze the role of Pedf on osteoblast function, we examined the effect of Pedf loss of function on osteoblast differentiation in vitro. In WT osteoblasts, Pedf mRNA expression and protein levels were low or undetectable immediately after plating but steadily increased during osteoblast differentiation with maximal levels evident at day 14 (Fig. 2A, B). The increased expression of Pedf at later times during differentiation suggests that the protein normally functions during matrix mineralization. Osteoblasts lacking Pedf appeared to differentiate relatively normally and had similar levels of alkaline phosphatase activity (Fig. 2C). However, matrix mineralization determined by Alizarin Red staining was increased when compared to cultures of WT osteoblasts (Fig. 2C) and FTIR measurements performed on mineralized nodules harvested at 14 days showed an increased mineral:matrix ratio of Pedf−/− cultures relative to that of WT cultures (Fig. 2D). The expression of several genes known to be involved in mineralization including annexins and Ank were unaffected in Pedf−/−osteoblasts whereas RT-PCR analysis revealed the levels of NPP1, Dmp1, Phex, and Bsp were reduced relative to WT controls (Fig. 2E, F).

image

Figure 2. Pedf expression and loss of function in primary cultured osteoblasts. (A) Relative expression levels of mRNA in samples collected in freshly plated osteoblasts (day 0) and at 7 and 14 days of culture in differentiation medium. (B) Western blot of Pedf protein levels in osteoblasts from wild-type (WT) mice at the indicated times in culture. Actin used as loading control. (C) Examination of osteoblast differentiation in WT and Pedf−/− osteoblasts. Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining, after 7 and 14 days of differentiation, respectively. (D) Mineral:matrix ratio of cultured osteoblasts. (E) RT-PCR analysis of Ank, NPP1, and Annexins 2, 5, and 6 expression performed at 14 days of differentiation. (F) RT-PCR analysis of Dmp1, Phex, and Bsp expression at day 14. All error bars represent SEM. *p < 0.05.

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Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

In this study, we characterized a mouse lacking Pedf, the protein product of a gene recently identified to be defective in patients with OI type VI.[10-13] The bone abnormalities in this mouse model closely resemble those seen in patients with OI type VI. The principal defect in bone from Pedf−/− mice was the excessive accumulation of unmineralized bone matrix, a phenotype also seen in patients with OI type VI. Moreover, FTIR analysis performed in histological sections from mouse femur and in primary mineralizing osteoblasts suggests that this matrix with an increased mineral to matrix ratio is abnormal, as in other types of OI.[28] Because preliminary analyses indicate the mineral composition is comparable to that in controls (data not shown), it appears that the matrix formed by mutant osteoblasts is unable to support normal mineralization. These features presumably account for the brittle nature of the bones that are more susceptible to fracture. However, precisely how loss of Pedf function causes the abnormal mineral-matrix composition in the mouse or in patients with OI type VI is still unclear. The reduced expression of several genes involved in bone mineralization in osteoblasts lacking Pedf is consistent with a role for the protein in matrix mineralization. In particular, mouse models with altered expression of Npp1, Dmp1, Phex, or Bsp exhibit defects in bone mineralization.[32-35] It is also possible that the ability of Pedf to control the degree and timing of vascular invasion in developing bone impacts osteoblast development and thereby contributes to the proper accumulation of bone matrix and deposition of mineral. Further studies to explore these potential functions are currently underway and will be facilitated by the mouse models described here.

Another important question relates to the mode of action of Pedf, and in particular whether Pedf is functioning in a cell (osteoblast) autonomous or cell nonautonomous fashion. Pedf is relatively widely expressed, is secreted into the circulation, and could certainly impact bone development through paracrine or hormonal mechanisms. Moreover, a number of Pedf receptors have been proposed, including ATGL/desnutrin/iPLA),[21] which are expressed in osteoblasts (data not shown). Alternatively, other studies suggest the possibility that Pedf might function in an intracrine mode through its ability to interact with transcription factors.[36, 37] These potential mechanisms will need to be elucidated using additional models in order to define precisely how Pedf functions in the production of bone matrix to ensure proper development and mineralization of bone.

In summary, the studies presented here confirm Pedf as a critical factor that controls the proper development of bone and ensures its structural integrity by controlling the deposition and mineralization of matrix. Moreover, the mouse model lacking Pedf will continue to provide a very powerful means to elucidate the mechanism through which PEDF impacts bone mineralization and integrity, and causes OI type VI. We predict that the discoveries from such experiments will inform research into more effective treatments for multiple skeletal disorders with mineralization defects.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

This work was supported by NIH grants AR049410 to TLC and AR043125 to AB. RCR is the recipient of a Career Development Award from the Veteran's Administration. TLC is a recipient of a Merit Review grant and a research career scientist award from the Veteran's Administration. We thank Vandana Singhal for technical assistance and Dr. Douglas DiGirolamo for advice in preparing the manuscript.

Authors' roles: Study design: RB, RCR, and TLC. Data collection: RB, RCR, ZL, SK, AN, MF, AB, and SEC. Data analysis: RB, RCR, AB, and TLC. Data interpretation: RB, RCR, AB, and TLC. Drafting manuscript: RB, RCR, and TLC. Revising manuscript content: RCR and TLC. Approving final version of manuscript: RB, RCR, ZL, SK, AN, MF, AB, SEC, and TLC. TLC takes responsibility for the integrity of the data analyses.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
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