- Email: [email protected]
Matrix metalloproteinase activities in avian tibial dyschondroplasia
PHYSIOLOGY AND REPRODUCTION Matrix Metalloproteinase Activities in Avian Tibial Dyschondroplasia N. C. RATH,* W. E. HUFF,* J. M. BALOG,* G. R. BAYYARI,* and R. P. REDDY†,1 *USDA/Agricultural Research Service, University of Arkansas, Fayetteville, Arkansas 72701, and †Peterson Farms Inc., Decatur, Arkansas 72722 kDa. On treatment with 4-aminophenylmercuric acetate, a compound that converts proenzyme forms of MMP, the 63 kDa MW gelatinolytic band migrated as a ∼60 kDa band and contributed to the broadening of the 59 kDa band. The TD-growth plate-conditioned media had significantly lower collagenolytic-gelatinolytic activities. The sulfated glycosaminoglycans, but not the collagen contents of the conditioned media from TD-explant cultures, were also reduced significantly. It is likely that the decreased matrix metalloproteinase activities of growth plate chondrocyte may contribute to a reduced turnover of extracellular matrices (ECM), leading to the retention of cartilage and its lack of vascularity in TDaffected growth plates.
(Key words: tibial dyschondroplasia, matrix metalloproteinase, conditioned media, glycosaminoglycans, collagen) 1997 Poultry Science 76:501–505
capable of complete degradation of matrical components (Woessner, 1995). The MMP are secreted by a variety of cells such as the fibroblasts, chondrocytes, macrophages, and endothelial cells that are present in the growth plate and their production is regulated by several autocrine and paracrine factors that facilitate connective tissue remodeling (Ogden et al., 1987; Ries and Petrides, 1995; Woessner, 1995). The MMP includes collagenase, gelatinase, and stromelysins, some of which are secreted as proenzymes in association with their endogenous inhibitors (Matrisian, 1992). Gelatin substrate zymography is a useful method to identify MMP of different molecular weights whose in vivo physiological substrate specificity may differ or overlap (Alexander and Werb, 1991; Matrisian, 1992; Ries and Petrides, 1995). Any abnormal regulation of these enzymes is likely to lead to pathological changes in connective tissue that may result in abnormal loss of matrix or lack of remodeling, and in vascularization. It is not known whether the impairment of matrix resorption in TD is related to some defective regulation in MMP of the growth plate. The physiological microenvironment of tissues, which includes their metabolic and secretory activities, can be studied in conditioned media (CM) prepared in vitro using cells or explant cultures. We have used media conditioned with cartilage explants derived from normal and TD-affected epiphyseal growth plates of chickens to study the gelatinase/matrix metalloproteinase activities and the
INTRODUCTION Tibial dyschondroplasia (TD) in poultry is a metabolic defect of the growth plate that results in the retention of a plug of cartilage that fails to resorb and undergo endochondral bone formation (Leach and Lilburn, 1992; Orth and Cook, 1994; Rath et al., 1994). This defect is common in adolescent meat-type poultry that are undergoing rapid growth. Although the etiology of TD is poorly understood, it is clear that the resorption of cartilage, its remodeling, and vascularization are impaired. Degradations of extracellular matrices (ECM) are an integral feature of connective tissue remodeling, cellular migration, and vascularization (Alexander and Werb, 1991). The growth plate, in adolescent birds and mammals, undergoes fast remodeling, resorption, and vascularization that lend to endochondral bone formation and long bone growth (Ogden et al., 1987). Collagens and proteoglycans are the most abundant extracellular matrix components of the epiphysis. Their degradations are mediated by a closely related family of endoproteinases called matrix metalloproteinases (MMP), which, in concert with other proteinases, are
Received for publication June 20, 1996. Accepted for publication October 18, 1996. 1Deceased.
501
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
ABSTRACT Tibial dyschondroplasia (TD) in poultry is a disorder of growth plate cartilage that fails to resorb and consequently prevents bone formation. Matrix metalloproteinases (MMP) contribute to the process of resorption through the degradation of extracellular matrices and facilitating vascularization, growth plate remodeling, and maturation. In order to understand the cause of the failure of cartilage degradation in TD, the gelatinase and collagenase activities, and the levels of collagen and glycosaminoglycans of conditioned media derived from cartilage-explant cultures of normal and TD growth plates were measured. Substrate zymography exhibited two prominent gelatinolytic and collagenolytic bands corresponding to MW 63, 59, and a broad but fuzzy band of activity between 100 and 200
502
RATH ET AL.
collagen, and proteoglycan contents to understand the mechanisms that may relate to the pathogenesis of TD.
MATERIALS AND METHODS
Tissues
Protein Determination The total protein content of the conditioned media and homogenate were determined using the Bradford dye binding assay.4 When necessary, the protein content of conditioned media from different birds was adjusted to an identical concentration using DMEM/F-12 culture media, and blank medium was used as a control for all determinations.
Determination of Collagenase The collagenase activities were determined by the ability of the conditioned media to digest Type IV collagen. The method used was a modification of the procedure described by Nethery et al., (1986). Briefly, Type IV human placental collagen was dissolved in 0.2% acetic acid and diluted to a concentration of 0.64 mg/mL in 100 mM Tris-HCl/200 mM NaCl buffer, pH 7.8 and coated onto 96-well micro titer plates5 at a volume of 50 mL (16 mg), incubated at 37 C in a humidified chamber for 24 h, followed by drying at 37 C overnight. The wells were
2Lixi Inc., Downers Grove, IL 60515. 3Celox Corp., Hopkins, MN 55343. 4BioRad, Hercules, CA 94547. 5Corning-Costar Corp., Cambridge, MA 02140. 6Polyscience, Warrington, PA 18976. 7BioTek Instruments, Winooski, VT 05404. 8Novex, San Diego, CA 92121.
[1 – (OD of sample wells/OD of control wells)] × 100. Cumulative results from seven samples in each group of normal and TD-affected chickens were analyzed using Student’s t test (SAS Institute, 1988).
Substrate Zymography Gelatin and collagen IV impregnated substratezymography was carried out according to Herron et al. (1986). An 11% sodium dodecyl sulfate-polyacrylamide slab gel was impregnated with gelatin or Type IV human placental collagen at a concentration of 0.1% prepared according to Laemmli (1970). Conditioned media diluted to an identical quantity of protein was mixed with 2× concentrations of nonreducing sample buffers (Laemmli, 1970) and electrophoresed at 100 constant voltages, 35 mA for ∼1.5 h in a Mini Protean II slab gel system.3 In some experiments, preincubation with 4-APMA was done as above for 10 min to 1.5 h prior to dilution with sample buffers before electrophoresis. The gels were washed for 30 min with 2.5% Triton X-100 with two successive changes and rinsed briefly in a collagenase buffer (50 mM Tris HCl, 5 mM CaCl2, 100 mM NaCl, 0.02% Na azide, pH 7.4), and incubated in the same buffer for 6 to 24 h at 37 or 23 C. For longer incubation periods, room temperature (23 C) was preferred, as it slowed down the reaction and enabled better distinction of bands. Inhibitors of different protease specificity were spiked into a collagenase buffer prior to the incubation of gels. The gels were stained in Coomassie brilliant blue and destained according to standard procedures. A prestained molecular weight standard8 was used to determine the approximate molecular weights of gelatinolytic components. The inhibitory ability of ethylenediaminetetraacetic acid (EDTA, a divalent cation chelator and matrix metal-
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
Normal and TD cartilages were obtained from 6-wk-old broiler chickens that were raised in floor pens with an NRC recommended diet (NRC, 1994) from Day 1 of age. Chickens were screened at 3 wk for the presence of TD using a Lixiscope2 and raised in separate pens thereafter at a density of 10 birds per pen. The chickens were killed at 6 wk of age using a guillotine, following which the cartilages were scraped from normal and TDaffected tibial growth plates under sterile conditions using a 1.5-mm curette. Individual growth plate-explants ∼1.5 to 2 mm thick, and weighing approximately 300 to 400 mg, were rinsed briefly in 5 mL of Dulbecco’s Modified Eagle’s Medium containing Ham’s F-12 salt mixture (DMEM/ F-12)3 and antibiotics (100 U penicillin, 100 mg streptomycin, and 0.25 mg amphotericin B/mL). The explants were then incubated in 1.5 mL of the same medium in a 24-well plate in a humidified incubator with 95% air and 5% CO2 for 24 h at 37 C. The conditioned media were centrifuged at 14,000 × g at 4 C and the supernatant stored at –20 C for further analyses.
rinsed with 250 mL distilled water twice for 2 min and the conditioned media diluted to a concentration of 10 mg of protein in 100 mL of buffer containing 0.1M NaCl, 50 mM Tris HCl, 5 mM CaCl2, 0.02% Na azide, pH 7.5. Because certain proenzyme forms of MMP are converted to active forms by 4-aminophenylmercuricacetate (APMA; Alexander and Werb, 1991), conditioned medium was activated by incubating in a final concentration of 2 mM APMA in 1% dimethyl sulfoxide for 1 h at 37 C then diluted to similar protein concentrations. Samples were applied in duplicate (50 mL, 10 mg protein) to collagencoated wells and incubated for 24 h. At the end of incubation, the wells were rinsed twice with distilled water, 5 min each, and reacted for 1 h with 100 mL of 0.1% Sirius red6 in saturated picric acid, to quantify collagen (Walsh et al., 1992). The wells were washed twice with 300 mL of distilled water followed with 10 mM HCl 5 min each twice to remove nonspecific Picro-Sirius red binding to wells. The bound dye was eluted with 100 mL of 0.1M NaOH and read at 550 nm in a micro plate reader.7 Blank media was used as the control. The average of duplicate wells was used for calculations. The relative percentage of collagenolysis was calculated as follows:
METALLOPROTEINASE AND TIBIAL DYSCHONDROPLASIA
503
loproteinase inhibitor), phenylmethylsulfonylfluoride (PMSF, a serine protease inhibitor), and 1,10 phenathroline (a matrix metalloproteinase inhibitor) on gelatinolytic activities were tested by including these compounds in the incubation buffer to a final concentration of 10 mM with respect to buffer.
Determination of Collagen and Sulfated Glycosaminoglycan Contents
FIGURE 1. A typical gelatin substrate zymography of conditioned media derived from cartilage explants from three individual birds in each group. Ten microgram equivalents conditioned media-protein electrophoresed in each lane and the gels were incubated at 23 C for 24 h. N1 to N3 and TD1 to TD3 represent CM derived from individual birds with normal and tibial dyschondroplastic cartilages. Bl = Blank media alone, Mr = Molecular weights markers.
RESULTS The hydroxyproline, sulfated glycosaminoglycan contents, and collagenase activities of the conditioned media are shown in Table 1. There was a significant decrease in the glycosaminoglycan content in conditioned media derived from TD cartilages. However, the collagen content did not show any significant difference between the normal and TD tissues. A significantly lower collagenolytic activity of the TD-conditioned media was evident (Table 1). A typical comparison of MMP activities between normal and TD cartilage conditioned media by gelatin zymography is shown in Figure 1. Two prominent gelatinase activities corresponding to MW approximately 63 and 59 kDa, and a broad but a fuzzy band of activity between 100 and 200 kDa were evident. The conditioned media derived from TD cartilage had consistently lower gelatinolytic activities than normal conditioned media equalized to identical protein concentrations. The broad band of gelatinolytic activity did not show any qualitative difference between normal and TD cartilage, nor was it affected by treatment with APMA. On short-term treatment with APMA, the 63 kDa gelatinase was reduced to a ∼60 kDa band (Figure 2a) and after 1 h of treatment it contributed to the broadening of 59 kDa MW band. Inclusion of either EDTA or 1, 10 phenanthroline, divalent cation chelator, in the incubation media abolished the gelatinase activities of the CM that was not affected by PMSF. The
9Serva Feinbiochemica, 10Sigma Chemical Co.,
Heidelberg, Germany. St. Louis, MO 63178-9916.
gelatinase bands closely corresponded to Type IV collagenase bands (Figure 2b).
DISCUSSION Matrix metalloproteinases have been implicated in growth plate remodeling and calcification (MikuniTakagaki and Cheng, 1987; Brown et al., 1993). Our results show that both normal and TD cartilages have collagenase/gelatinase activities that may be involved in the resorption and turnover of extracellular matrices in the growth plate. The TD-affected cartilage explants however, showed significantly lower collagenasegelatinase content by quantitative methods and qualitative judgment of zymograms. The reduced biological activities’ MMP may relate to reduced resorption and remodeling of the growth plate cartilage. The chicken gelatinases have been isolated in conditioned media of skin fibroblasts (Craig et al., 1991), and from embryonic tibial explant cultures (Mikuni-Takagaki and Cheng, 1987; Cole et al., 1993). The tibial culture conditioned media have been shown to have both proteoglycan and Type X collagen degrading activities. Our results show the presence of two distinct bands of gelatinase in the growth plate explant CM that correspond to MW approximately 63 and 59 kDa and a broad but fuzzy band between 100 and 200 kDa. These MW are in general agreement with those reported in the literature (Mikuni-Takagaki and Cheng, 1987; Craig et al., 1991; Cole et al., 1993). The gelatinolytic bands also corresponded to the collagenolytic bands observed using
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
The conditioned medium was hydrolyzed in 6N HCl at 110 C for 24 h for the determination of hydroxyproline content of the samples according to a micro titer plate assay method of Cawston et al. (1994). The hydroxyproline data was converted to collagen by multiplying each value by 7.46 based on the assumption that 13% of collagen is hydroxyproline. Sulfated glycosaminoglycan content was estimated by the 1,9-dimethyl methylene blue9 dye binding procedure as described by Chandrasekhar et al. (1987) using chondroitin 4-sulfate as the standard. The quantitative results were compared using Student’s t test (SAS Institute, 1988). All chemicals were purchased from Sigma Chemical Co.10 unless otherwise stated.
504
RATH ET AL. TABLE 1. Collagen, sulfated glycosaminoglycan, and collagenolytic activities of conditioned media derived from normal and tibial dyschondroplastic (TD) cartilage explants (Mean ± SEM, n = 7) Growth plate explants
Collagen
Normal TD
0.198 ± 0.017 0.226 ± 0.035
Sulfated glycosaminoglycans (mg/mg protein) 5.49 ± 0.83 1.06 ± 0.25*
Collagenolytic activity (% lysis) 49.14 ± 3.57 22.26 ± 2.91*
*P ≤ 0.005.
FIGURE 2. Zymographic visualization of the effect of 4aminophenylmercuricacetate (APMA) on conditioned media MMP using 10 mg of protein electrophoresed on (a) gelatin substrate-gel and (b) type IV collagen substrate-gel. Gels were developed 16 h at 23 C. (a) lane 1 = MW marker; lane 2 = CM without APMA treatment; lane 3 = CM with 1 h treatment with APMA; lane 4 = CM with 10 min APMA treatment. (b) lane 1 = CM without APMA treatment, lane 2 = CM with 1 h treatment with APMA.
synthesis and degradation of proteoglycans in dystrophic cartilage plugs of chickens. The absence of any difference in the collagen content of conditioned media derived from normal and TD cartilage was intriguing. However, this discrepancy could also be due to the differences in the rate of turnover of different ECM components that has been observed using normal cartilage organ cultures, in which degradation of proteoglycans preceded collagen by several days (Cole et al., 1993). Therefore, if there was any difference in the collagen degradation between normal and TD-cartilage explants, it was not discernible during the 24 h periods of culture in our experiment. A variety of cytokines and growth factors regulate the expression and secretion of MMP by the skeletal tissues, which, in turn, may control the turnover of ECM and remodeling (Schnyder et al., 1987; Smith et al., 1989). Fibroblast growth factors (FGF) for example, induces collagenase in skeletal tissues and facilitates angiogenesis (Ries and Petrides, 1995; Varghese et al., 1995), and TD-affected cartilage has been shown to be deficient in FGF (Twal et al., 1996). On the other hand, the ability of TD-affected chondrocytes to respond to these regulatory cytokines and growth factors may be only a part of this problem. Studies from other laboratories have demonstrated that active resorption of cartilage matrices requires the presence of viable chondrocytes and vascular cells (Gibson et al., 1995). Recent data from our laboratory showed an extensive apoptosis occurring in TD-affected cartilages (Rath et al., 1996) that may secondarily affect an overall decrease in cartilage metabolism of the TD growth plate. The cartilage extracellular matrix may contain factors that prevent invasiveness of endothelial cells and capillaries; these factors can be removed through degradation of ECM (Kuettner and Pauli, 1983; Moses et al., 1992). Similarly, degradation of ECM may cause the release of matrix-bound activating factors that would promote angiogenesis (Hirshman and Dziewiatkowski, 1966; Brown et al., 1993; Fisher et al., 1994). In the absence of matrix degradation, the persistence of cartilage is likely to occur. In conclusion, our results indicate that reduced levels of ECM degrading MMP in TD growth plates may be partly responsible for the occurrence of this defect.
ACKNOWLEDGMENTS The authors wish to thank David Horlick, Scott Zornes, and Dana Bassi for excellent technical assistance
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
Type IV collagen as substrate. The 63 kDa MW gelatinase was reduced to an intermediate 60 kDa MW band on short term treatment with 4-APMA and contributed to the broadening of the 59 kDa band at 1 h of activation. The 59 kDa band may be the activated form of the 63 kDa band gelatinase. The susceptibility of the gelatinase to inhibition by EDTA, and 1, 10 phenanthroline is a characteristic feature of metalloproteinases. Although the exact function of gelatinases in the metabolism of different ECM components in the growth plate remains to be understood, it is known to regulate remodeling, vascularization, and calcification of cartilage in concert with other proteinases such as cathepsin (Mikuni-Takagaki and Cheng, 1987; Blair et al., 1989; Matrisian, 1992; Brown et al., 1993; Sires et al., 1995). It is possible that a down-regulation of MMP may contribute to a reduced resorption of ECM, failure of vascularization, and to the persistence of the cartilage in TD. This effect was also evident by a reduction of sulfated glycosaminoglycans in TD-cartilage-conditioned media. Lowther et al. (1974) observed both decreased
METALLOPROTEINASE AND TIBIAL DYSCHONDROPLASIA
and D. Barnes and S. Chandrasekhar for helpful suggestions.
REFERENCES
Mikuni-Takagaki, Y., and Y. S. Cheng, 1987. Metalloproteinases in endochondral bone formation; appearance of tissue inhibitor-resistant metalloproteinases. Arch. Biochem. Biophys. 259:576–588. Moses, M. A., J. Sudhalter, and R. Langer, 1992. Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes. J. Cell Biol. 119:475–482. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Nethery, A., J. G. Lyons, and R. L. O’Grady, 1986. A spectrophotometric collagenase assay. Anal. Biochem. 159: 390–395. Ogden, J. A., D. P. Grogan, and T. A. Light, 1987. Postnatal development and growth of the musculoskeletal system. Pages 91–160 in: The Scientific Basis of Orthopaedics. J. A. Albright and R. A. Brand, ed. Appleton and Lang Publishers, Norwalk, CT. Orth, M. W., and M. E. Cook, 1994. Avian tibial dyschondroplasia: a morphological and biochemical review of the growth plate lesion and its causes. Vet. Pathol. 31:403–414. Rath, N. C., G. R. Bayyari, J. M. Balog, and W. E. Huff, 1994. Physiological studies of turkey tibial dyschondroplasia. Poultry Sci. 73:416–424. Rath, N. C., W. E. Huff, J. M. Balog, and G. R. Bayyari, 1996. Biochemical and cellular manifestations of epiphyseal growth plate in avian tibial dyschondroplasia. Pages 132–133 in: Proceedings of the 45th Western Poultry Disease Conference, Cancun, Mexico. Ries, C., and P. E. Petrides, 1995. Cytokine regulation of matrix metalloproteinase activity and its regulatory dysfunction in disease. Biol. Chem. Hoppe-Seyler. 376:345–355. SAS Institute, 1988. SAS Users Guide: Statistics. 1988 ed. SAS Institute Inc., Cary, NC. Schnyder, J., T. Payne, and C. Dinarello, 1987. Human monocyte or recombinant interleukin-1’s are specific for the secretion of a metalloproteinase from chondrocytes. J. Immunol. 138:496–503. Sires, U. I., T. M. Schimd, C. J. Fliszar, Z. Q. Wang, and S. L. Gluck, 1995. Complete degradation of type X collagen requires combined action of interstitial collagenase and osteoclast-derived cathepsin-B. J. Clin. Invest. 95: 2089–2095. Smith, R. J., N. A. Rohloff, L. M. Sam, J. M. Justen, M. R. Diebel, and J. C. Cornette, 1989. Recombinant human interleukin-a and recombinant human interleukin-b stimulate cartilage matrix degradation and inhibit glycosaminoglycan synthesis. Inflammation 13:367–382. Twal, W. O., J. Wu, C. V. Gay, and R. M. Leach, Jr., 1996. Immunolocalization of basic fibroblast growth factor in avian tibial dyschondroplasia. Poultry Sci. 75:130–134. Varghese, S., M. L. Ramsby, and E. Canalis, 1995. Basic fibroblast growth factor stimulates expression of interstitial collagenase and inhibitors of metalloproteinases in rat bone cells. Endocrinology 136:2156–2162. Walsh, B., S. C. Thornton, R. Penny, and S. N. Breit, 1992. Micro plate reader-based quantitation of collagens. Anal. Biochem. 203:187–190. Woessner, J. F., Jr. 1995. Quantification of matrixmetalloproteinases in tissue samples. Methods Enzymol. 248:510–531.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
Alexander, C. M., and Z. Werb, 1991. Extracellular matrix degradation. Pages 255–302 in: Cell Biology of Extracellular Matrix. E. D. Hay, ed. Plenum Press, New York, NY. Blair, H. C., D. D. Dean, D. S. Howell, S. L. Teitelbaum, and J. J. Jeffery, 1989. Hypertrophic chondrocytes produce immunoreactive collagenase in vivo. Connective Tissue Res. 23:65–73. Brown, R. A., M. Kayser, B. McLaughlin, and J. B. Weiss, 1993. Collagenase and gelatinase production by calcifying growth plate chondrocytes. Exp. Cell Res. 208:1–9. Cawston, T., T. Plumpton, V. Curry, A. Ellis, and L. Powell, 1994. Role of TIMP and MMP inhibition in preventing connective tissue breakdown. Ann. N.Y. Acad. Sci. 732: 75–83. Chandrasekhar, S., M. Esterman, and H. A. Hoffman, 1987. Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal. Biochem. 161:103–108. Cole, A., T. Boyd, L. Luchene, K. E. Kuettner, and T. M. Schmid, 1993. Type X collagen degradation in long term serum-free culture of the embryonic chick tibia following production of active collagenase and gelatinase. Dev. Biol. 159:528–534. Craig, F. M., C. W. Archer, and G. Murphy, 1991. Isolation and characterization of a chicken gelatinase (type IV collagenase). Biochim. Biophys. Acta 1074:243–250. Fisher, C., S. Gilbertson-Beadling, E. A. Powers, G. Petzold, R. Poorman, and M. A. Mitchell, 1994. Interstitial collagenase is required for angiogenesis in vitro. Dev. Biol. 162: 499–510. Gibson, G. J., D. L. Lin, M. B. Schaffler, and J. H. Kimura, 1995. Endochondral resorption of chicken sterna in culture. J. Orthop. Res. 13:542–552. Herron, G. S., M. J. Banda, E. J. Clark, J. Gravilovic, and Z. Werb, 1986. Secretion of matrix metalloproteinase by capillary endothelial cells II. expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. J. Biol. Chem. 261:2814–2818. Hirshman, A., and D. D. Dziewiatkowski, 1966. Protein polysaccharide loss during endochondral ossification: Immunochemical evidence. Science 154:393–395. Kuettner, K., and B. U. Pauli, 1983. Vascularity of cartilage. Pages 281–308 in: Cartilage. Volume I. B. K. Hall, ed. Academic Press, New York, NY. Laemmli, U. K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227:680–685. Leach, R. M., and M. S. Lilburn, 1992. Current knowledge on the etiology of tibial dyschondroplasia in the avian species. Poult. Sci. Rev. 4:57–65. Lowther, D. A., H. C. Robinson, J. W. Dolman, and K. W. Thomas, 1974. Cartilage matrix components in chickens with tibial dyschondroplasia. J. Nutr. 104:922–929. Matrisian, I. M., 1992. The matrix-degrading metalloproteinases. Bioassays 14:455–463.
505
(Key words: tibial dyschondroplasia, matrix metalloproteinase, conditioned media, glycosaminoglycans, collagen) 1997 Poultry Science 76:501–505
capable of complete degradation of matrical components (Woessner, 1995). The MMP are secreted by a variety of cells such as the fibroblasts, chondrocytes, macrophages, and endothelial cells that are present in the growth plate and their production is regulated by several autocrine and paracrine factors that facilitate connective tissue remodeling (Ogden et al., 1987; Ries and Petrides, 1995; Woessner, 1995). The MMP includes collagenase, gelatinase, and stromelysins, some of which are secreted as proenzymes in association with their endogenous inhibitors (Matrisian, 1992). Gelatin substrate zymography is a useful method to identify MMP of different molecular weights whose in vivo physiological substrate specificity may differ or overlap (Alexander and Werb, 1991; Matrisian, 1992; Ries and Petrides, 1995). Any abnormal regulation of these enzymes is likely to lead to pathological changes in connective tissue that may result in abnormal loss of matrix or lack of remodeling, and in vascularization. It is not known whether the impairment of matrix resorption in TD is related to some defective regulation in MMP of the growth plate. The physiological microenvironment of tissues, which includes their metabolic and secretory activities, can be studied in conditioned media (CM) prepared in vitro using cells or explant cultures. We have used media conditioned with cartilage explants derived from normal and TD-affected epiphyseal growth plates of chickens to study the gelatinase/matrix metalloproteinase activities and the
INTRODUCTION Tibial dyschondroplasia (TD) in poultry is a metabolic defect of the growth plate that results in the retention of a plug of cartilage that fails to resorb and undergo endochondral bone formation (Leach and Lilburn, 1992; Orth and Cook, 1994; Rath et al., 1994). This defect is common in adolescent meat-type poultry that are undergoing rapid growth. Although the etiology of TD is poorly understood, it is clear that the resorption of cartilage, its remodeling, and vascularization are impaired. Degradations of extracellular matrices (ECM) are an integral feature of connective tissue remodeling, cellular migration, and vascularization (Alexander and Werb, 1991). The growth plate, in adolescent birds and mammals, undergoes fast remodeling, resorption, and vascularization that lend to endochondral bone formation and long bone growth (Ogden et al., 1987). Collagens and proteoglycans are the most abundant extracellular matrix components of the epiphysis. Their degradations are mediated by a closely related family of endoproteinases called matrix metalloproteinases (MMP), which, in concert with other proteinases, are
Received for publication June 20, 1996. Accepted for publication October 18, 1996. 1Deceased.
501
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
ABSTRACT Tibial dyschondroplasia (TD) in poultry is a disorder of growth plate cartilage that fails to resorb and consequently prevents bone formation. Matrix metalloproteinases (MMP) contribute to the process of resorption through the degradation of extracellular matrices and facilitating vascularization, growth plate remodeling, and maturation. In order to understand the cause of the failure of cartilage degradation in TD, the gelatinase and collagenase activities, and the levels of collagen and glycosaminoglycans of conditioned media derived from cartilage-explant cultures of normal and TD growth plates were measured. Substrate zymography exhibited two prominent gelatinolytic and collagenolytic bands corresponding to MW 63, 59, and a broad but fuzzy band of activity between 100 and 200
502
RATH ET AL.
collagen, and proteoglycan contents to understand the mechanisms that may relate to the pathogenesis of TD.
MATERIALS AND METHODS
Tissues
Protein Determination The total protein content of the conditioned media and homogenate were determined using the Bradford dye binding assay.4 When necessary, the protein content of conditioned media from different birds was adjusted to an identical concentration using DMEM/F-12 culture media, and blank medium was used as a control for all determinations.
Determination of Collagenase The collagenase activities were determined by the ability of the conditioned media to digest Type IV collagen. The method used was a modification of the procedure described by Nethery et al., (1986). Briefly, Type IV human placental collagen was dissolved in 0.2% acetic acid and diluted to a concentration of 0.64 mg/mL in 100 mM Tris-HCl/200 mM NaCl buffer, pH 7.8 and coated onto 96-well micro titer plates5 at a volume of 50 mL (16 mg), incubated at 37 C in a humidified chamber for 24 h, followed by drying at 37 C overnight. The wells were
2Lixi Inc., Downers Grove, IL 60515. 3Celox Corp., Hopkins, MN 55343. 4BioRad, Hercules, CA 94547. 5Corning-Costar Corp., Cambridge, MA 02140. 6Polyscience, Warrington, PA 18976. 7BioTek Instruments, Winooski, VT 05404. 8Novex, San Diego, CA 92121.
[1 – (OD of sample wells/OD of control wells)] × 100. Cumulative results from seven samples in each group of normal and TD-affected chickens were analyzed using Student’s t test (SAS Institute, 1988).
Substrate Zymography Gelatin and collagen IV impregnated substratezymography was carried out according to Herron et al. (1986). An 11% sodium dodecyl sulfate-polyacrylamide slab gel was impregnated with gelatin or Type IV human placental collagen at a concentration of 0.1% prepared according to Laemmli (1970). Conditioned media diluted to an identical quantity of protein was mixed with 2× concentrations of nonreducing sample buffers (Laemmli, 1970) and electrophoresed at 100 constant voltages, 35 mA for ∼1.5 h in a Mini Protean II slab gel system.3 In some experiments, preincubation with 4-APMA was done as above for 10 min to 1.5 h prior to dilution with sample buffers before electrophoresis. The gels were washed for 30 min with 2.5% Triton X-100 with two successive changes and rinsed briefly in a collagenase buffer (50 mM Tris HCl, 5 mM CaCl2, 100 mM NaCl, 0.02% Na azide, pH 7.4), and incubated in the same buffer for 6 to 24 h at 37 or 23 C. For longer incubation periods, room temperature (23 C) was preferred, as it slowed down the reaction and enabled better distinction of bands. Inhibitors of different protease specificity were spiked into a collagenase buffer prior to the incubation of gels. The gels were stained in Coomassie brilliant blue and destained according to standard procedures. A prestained molecular weight standard8 was used to determine the approximate molecular weights of gelatinolytic components. The inhibitory ability of ethylenediaminetetraacetic acid (EDTA, a divalent cation chelator and matrix metal-
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
Normal and TD cartilages were obtained from 6-wk-old broiler chickens that were raised in floor pens with an NRC recommended diet (NRC, 1994) from Day 1 of age. Chickens were screened at 3 wk for the presence of TD using a Lixiscope2 and raised in separate pens thereafter at a density of 10 birds per pen. The chickens were killed at 6 wk of age using a guillotine, following which the cartilages were scraped from normal and TDaffected tibial growth plates under sterile conditions using a 1.5-mm curette. Individual growth plate-explants ∼1.5 to 2 mm thick, and weighing approximately 300 to 400 mg, were rinsed briefly in 5 mL of Dulbecco’s Modified Eagle’s Medium containing Ham’s F-12 salt mixture (DMEM/ F-12)3 and antibiotics (100 U penicillin, 100 mg streptomycin, and 0.25 mg amphotericin B/mL). The explants were then incubated in 1.5 mL of the same medium in a 24-well plate in a humidified incubator with 95% air and 5% CO2 for 24 h at 37 C. The conditioned media were centrifuged at 14,000 × g at 4 C and the supernatant stored at –20 C for further analyses.
rinsed with 250 mL distilled water twice for 2 min and the conditioned media diluted to a concentration of 10 mg of protein in 100 mL of buffer containing 0.1M NaCl, 50 mM Tris HCl, 5 mM CaCl2, 0.02% Na azide, pH 7.5. Because certain proenzyme forms of MMP are converted to active forms by 4-aminophenylmercuricacetate (APMA; Alexander and Werb, 1991), conditioned medium was activated by incubating in a final concentration of 2 mM APMA in 1% dimethyl sulfoxide for 1 h at 37 C then diluted to similar protein concentrations. Samples were applied in duplicate (50 mL, 10 mg protein) to collagencoated wells and incubated for 24 h. At the end of incubation, the wells were rinsed twice with distilled water, 5 min each, and reacted for 1 h with 100 mL of 0.1% Sirius red6 in saturated picric acid, to quantify collagen (Walsh et al., 1992). The wells were washed twice with 300 mL of distilled water followed with 10 mM HCl 5 min each twice to remove nonspecific Picro-Sirius red binding to wells. The bound dye was eluted with 100 mL of 0.1M NaOH and read at 550 nm in a micro plate reader.7 Blank media was used as the control. The average of duplicate wells was used for calculations. The relative percentage of collagenolysis was calculated as follows:
METALLOPROTEINASE AND TIBIAL DYSCHONDROPLASIA
503
loproteinase inhibitor), phenylmethylsulfonylfluoride (PMSF, a serine protease inhibitor), and 1,10 phenathroline (a matrix metalloproteinase inhibitor) on gelatinolytic activities were tested by including these compounds in the incubation buffer to a final concentration of 10 mM with respect to buffer.
Determination of Collagen and Sulfated Glycosaminoglycan Contents
FIGURE 1. A typical gelatin substrate zymography of conditioned media derived from cartilage explants from three individual birds in each group. Ten microgram equivalents conditioned media-protein electrophoresed in each lane and the gels were incubated at 23 C for 24 h. N1 to N3 and TD1 to TD3 represent CM derived from individual birds with normal and tibial dyschondroplastic cartilages. Bl = Blank media alone, Mr = Molecular weights markers.
RESULTS The hydroxyproline, sulfated glycosaminoglycan contents, and collagenase activities of the conditioned media are shown in Table 1. There was a significant decrease in the glycosaminoglycan content in conditioned media derived from TD cartilages. However, the collagen content did not show any significant difference between the normal and TD tissues. A significantly lower collagenolytic activity of the TD-conditioned media was evident (Table 1). A typical comparison of MMP activities between normal and TD cartilage conditioned media by gelatin zymography is shown in Figure 1. Two prominent gelatinase activities corresponding to MW approximately 63 and 59 kDa, and a broad but a fuzzy band of activity between 100 and 200 kDa were evident. The conditioned media derived from TD cartilage had consistently lower gelatinolytic activities than normal conditioned media equalized to identical protein concentrations. The broad band of gelatinolytic activity did not show any qualitative difference between normal and TD cartilage, nor was it affected by treatment with APMA. On short-term treatment with APMA, the 63 kDa gelatinase was reduced to a ∼60 kDa band (Figure 2a) and after 1 h of treatment it contributed to the broadening of 59 kDa MW band. Inclusion of either EDTA or 1, 10 phenanthroline, divalent cation chelator, in the incubation media abolished the gelatinase activities of the CM that was not affected by PMSF. The
9Serva Feinbiochemica, 10Sigma Chemical Co.,
Heidelberg, Germany. St. Louis, MO 63178-9916.
gelatinase bands closely corresponded to Type IV collagenase bands (Figure 2b).
DISCUSSION Matrix metalloproteinases have been implicated in growth plate remodeling and calcification (MikuniTakagaki and Cheng, 1987; Brown et al., 1993). Our results show that both normal and TD cartilages have collagenase/gelatinase activities that may be involved in the resorption and turnover of extracellular matrices in the growth plate. The TD-affected cartilage explants however, showed significantly lower collagenasegelatinase content by quantitative methods and qualitative judgment of zymograms. The reduced biological activities’ MMP may relate to reduced resorption and remodeling of the growth plate cartilage. The chicken gelatinases have been isolated in conditioned media of skin fibroblasts (Craig et al., 1991), and from embryonic tibial explant cultures (Mikuni-Takagaki and Cheng, 1987; Cole et al., 1993). The tibial culture conditioned media have been shown to have both proteoglycan and Type X collagen degrading activities. Our results show the presence of two distinct bands of gelatinase in the growth plate explant CM that correspond to MW approximately 63 and 59 kDa and a broad but fuzzy band between 100 and 200 kDa. These MW are in general agreement with those reported in the literature (Mikuni-Takagaki and Cheng, 1987; Craig et al., 1991; Cole et al., 1993). The gelatinolytic bands also corresponded to the collagenolytic bands observed using
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
The conditioned medium was hydrolyzed in 6N HCl at 110 C for 24 h for the determination of hydroxyproline content of the samples according to a micro titer plate assay method of Cawston et al. (1994). The hydroxyproline data was converted to collagen by multiplying each value by 7.46 based on the assumption that 13% of collagen is hydroxyproline. Sulfated glycosaminoglycan content was estimated by the 1,9-dimethyl methylene blue9 dye binding procedure as described by Chandrasekhar et al. (1987) using chondroitin 4-sulfate as the standard. The quantitative results were compared using Student’s t test (SAS Institute, 1988). All chemicals were purchased from Sigma Chemical Co.10 unless otherwise stated.
504
RATH ET AL. TABLE 1. Collagen, sulfated glycosaminoglycan, and collagenolytic activities of conditioned media derived from normal and tibial dyschondroplastic (TD) cartilage explants (Mean ± SEM, n = 7) Growth plate explants
Collagen
Normal TD
0.198 ± 0.017 0.226 ± 0.035
Sulfated glycosaminoglycans (mg/mg protein) 5.49 ± 0.83 1.06 ± 0.25*
Collagenolytic activity (% lysis) 49.14 ± 3.57 22.26 ± 2.91*
*P ≤ 0.005.
FIGURE 2. Zymographic visualization of the effect of 4aminophenylmercuricacetate (APMA) on conditioned media MMP using 10 mg of protein electrophoresed on (a) gelatin substrate-gel and (b) type IV collagen substrate-gel. Gels were developed 16 h at 23 C. (a) lane 1 = MW marker; lane 2 = CM without APMA treatment; lane 3 = CM with 1 h treatment with APMA; lane 4 = CM with 10 min APMA treatment. (b) lane 1 = CM without APMA treatment, lane 2 = CM with 1 h treatment with APMA.
synthesis and degradation of proteoglycans in dystrophic cartilage plugs of chickens. The absence of any difference in the collagen content of conditioned media derived from normal and TD cartilage was intriguing. However, this discrepancy could also be due to the differences in the rate of turnover of different ECM components that has been observed using normal cartilage organ cultures, in which degradation of proteoglycans preceded collagen by several days (Cole et al., 1993). Therefore, if there was any difference in the collagen degradation between normal and TD-cartilage explants, it was not discernible during the 24 h periods of culture in our experiment. A variety of cytokines and growth factors regulate the expression and secretion of MMP by the skeletal tissues, which, in turn, may control the turnover of ECM and remodeling (Schnyder et al., 1987; Smith et al., 1989). Fibroblast growth factors (FGF) for example, induces collagenase in skeletal tissues and facilitates angiogenesis (Ries and Petrides, 1995; Varghese et al., 1995), and TD-affected cartilage has been shown to be deficient in FGF (Twal et al., 1996). On the other hand, the ability of TD-affected chondrocytes to respond to these regulatory cytokines and growth factors may be only a part of this problem. Studies from other laboratories have demonstrated that active resorption of cartilage matrices requires the presence of viable chondrocytes and vascular cells (Gibson et al., 1995). Recent data from our laboratory showed an extensive apoptosis occurring in TD-affected cartilages (Rath et al., 1996) that may secondarily affect an overall decrease in cartilage metabolism of the TD growth plate. The cartilage extracellular matrix may contain factors that prevent invasiveness of endothelial cells and capillaries; these factors can be removed through degradation of ECM (Kuettner and Pauli, 1983; Moses et al., 1992). Similarly, degradation of ECM may cause the release of matrix-bound activating factors that would promote angiogenesis (Hirshman and Dziewiatkowski, 1966; Brown et al., 1993; Fisher et al., 1994). In the absence of matrix degradation, the persistence of cartilage is likely to occur. In conclusion, our results indicate that reduced levels of ECM degrading MMP in TD growth plates may be partly responsible for the occurrence of this defect.
ACKNOWLEDGMENTS The authors wish to thank David Horlick, Scott Zornes, and Dana Bassi for excellent technical assistance
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
Type IV collagen as substrate. The 63 kDa MW gelatinase was reduced to an intermediate 60 kDa MW band on short term treatment with 4-APMA and contributed to the broadening of the 59 kDa band at 1 h of activation. The 59 kDa band may be the activated form of the 63 kDa band gelatinase. The susceptibility of the gelatinase to inhibition by EDTA, and 1, 10 phenanthroline is a characteristic feature of metalloproteinases. Although the exact function of gelatinases in the metabolism of different ECM components in the growth plate remains to be understood, it is known to regulate remodeling, vascularization, and calcification of cartilage in concert with other proteinases such as cathepsin (Mikuni-Takagaki and Cheng, 1987; Blair et al., 1989; Matrisian, 1992; Brown et al., 1993; Sires et al., 1995). It is possible that a down-regulation of MMP may contribute to a reduced resorption of ECM, failure of vascularization, and to the persistence of the cartilage in TD. This effect was also evident by a reduction of sulfated glycosaminoglycans in TD-cartilage-conditioned media. Lowther et al. (1974) observed both decreased
METALLOPROTEINASE AND TIBIAL DYSCHONDROPLASIA
and D. Barnes and S. Chandrasekhar for helpful suggestions.
REFERENCES
Mikuni-Takagaki, Y., and Y. S. Cheng, 1987. Metalloproteinases in endochondral bone formation; appearance of tissue inhibitor-resistant metalloproteinases. Arch. Biochem. Biophys. 259:576–588. Moses, M. A., J. Sudhalter, and R. Langer, 1992. Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes. J. Cell Biol. 119:475–482. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Nethery, A., J. G. Lyons, and R. L. O’Grady, 1986. A spectrophotometric collagenase assay. Anal. Biochem. 159: 390–395. Ogden, J. A., D. P. Grogan, and T. A. Light, 1987. Postnatal development and growth of the musculoskeletal system. Pages 91–160 in: The Scientific Basis of Orthopaedics. J. A. Albright and R. A. Brand, ed. Appleton and Lang Publishers, Norwalk, CT. Orth, M. W., and M. E. Cook, 1994. Avian tibial dyschondroplasia: a morphological and biochemical review of the growth plate lesion and its causes. Vet. Pathol. 31:403–414. Rath, N. C., G. R. Bayyari, J. M. Balog, and W. E. Huff, 1994. Physiological studies of turkey tibial dyschondroplasia. Poultry Sci. 73:416–424. Rath, N. C., W. E. Huff, J. M. Balog, and G. R. Bayyari, 1996. Biochemical and cellular manifestations of epiphyseal growth plate in avian tibial dyschondroplasia. Pages 132–133 in: Proceedings of the 45th Western Poultry Disease Conference, Cancun, Mexico. Ries, C., and P. E. Petrides, 1995. Cytokine regulation of matrix metalloproteinase activity and its regulatory dysfunction in disease. Biol. Chem. Hoppe-Seyler. 376:345–355. SAS Institute, 1988. SAS Users Guide: Statistics. 1988 ed. SAS Institute Inc., Cary, NC. Schnyder, J., T. Payne, and C. Dinarello, 1987. Human monocyte or recombinant interleukin-1’s are specific for the secretion of a metalloproteinase from chondrocytes. J. Immunol. 138:496–503. Sires, U. I., T. M. Schimd, C. J. Fliszar, Z. Q. Wang, and S. L. Gluck, 1995. Complete degradation of type X collagen requires combined action of interstitial collagenase and osteoclast-derived cathepsin-B. J. Clin. Invest. 95: 2089–2095. Smith, R. J., N. A. Rohloff, L. M. Sam, J. M. Justen, M. R. Diebel, and J. C. Cornette, 1989. Recombinant human interleukin-a and recombinant human interleukin-b stimulate cartilage matrix degradation and inhibit glycosaminoglycan synthesis. Inflammation 13:367–382. Twal, W. O., J. Wu, C. V. Gay, and R. M. Leach, Jr., 1996. Immunolocalization of basic fibroblast growth factor in avian tibial dyschondroplasia. Poultry Sci. 75:130–134. Varghese, S., M. L. Ramsby, and E. Canalis, 1995. Basic fibroblast growth factor stimulates expression of interstitial collagenase and inhibitors of metalloproteinases in rat bone cells. Endocrinology 136:2156–2162. Walsh, B., S. C. Thornton, R. Penny, and S. N. Breit, 1992. Micro plate reader-based quantitation of collagens. Anal. Biochem. 203:187–190. Woessner, J. F., Jr. 1995. Quantification of matrixmetalloproteinases in tissue samples. Methods Enzymol. 248:510–531.
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on April 21, 2015
Alexander, C. M., and Z. Werb, 1991. Extracellular matrix degradation. Pages 255–302 in: Cell Biology of Extracellular Matrix. E. D. Hay, ed. Plenum Press, New York, NY. Blair, H. C., D. D. Dean, D. S. Howell, S. L. Teitelbaum, and J. J. Jeffery, 1989. Hypertrophic chondrocytes produce immunoreactive collagenase in vivo. Connective Tissue Res. 23:65–73. Brown, R. A., M. Kayser, B. McLaughlin, and J. B. Weiss, 1993. Collagenase and gelatinase production by calcifying growth plate chondrocytes. Exp. Cell Res. 208:1–9. Cawston, T., T. Plumpton, V. Curry, A. Ellis, and L. Powell, 1994. Role of TIMP and MMP inhibition in preventing connective tissue breakdown. Ann. N.Y. Acad. Sci. 732: 75–83. Chandrasekhar, S., M. Esterman, and H. A. Hoffman, 1987. Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal. Biochem. 161:103–108. Cole, A., T. Boyd, L. Luchene, K. E. Kuettner, and T. M. Schmid, 1993. Type X collagen degradation in long term serum-free culture of the embryonic chick tibia following production of active collagenase and gelatinase. Dev. Biol. 159:528–534. Craig, F. M., C. W. Archer, and G. Murphy, 1991. Isolation and characterization of a chicken gelatinase (type IV collagenase). Biochim. Biophys. Acta 1074:243–250. Fisher, C., S. Gilbertson-Beadling, E. A. Powers, G. Petzold, R. Poorman, and M. A. Mitchell, 1994. Interstitial collagenase is required for angiogenesis in vitro. Dev. Biol. 162: 499–510. Gibson, G. J., D. L. Lin, M. B. Schaffler, and J. H. Kimura, 1995. Endochondral resorption of chicken sterna in culture. J. Orthop. Res. 13:542–552. Herron, G. S., M. J. Banda, E. J. Clark, J. Gravilovic, and Z. Werb, 1986. Secretion of matrix metalloproteinase by capillary endothelial cells II. expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. J. Biol. Chem. 261:2814–2818. Hirshman, A., and D. D. Dziewiatkowski, 1966. Protein polysaccharide loss during endochondral ossification: Immunochemical evidence. Science 154:393–395. Kuettner, K., and B. U. Pauli, 1983. Vascularity of cartilage. Pages 281–308 in: Cartilage. Volume I. B. K. Hall, ed. Academic Press, New York, NY. Laemmli, U. K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227:680–685. Leach, R. M., and M. S. Lilburn, 1992. Current knowledge on the etiology of tibial dyschondroplasia in the avian species. Poult. Sci. Rev. 4:57–65. Lowther, D. A., H. C. Robinson, J. W. Dolman, and K. W. Thomas, 1974. Cartilage matrix components in chickens with tibial dyschondroplasia. J. Nutr. 104:922–929. Matrisian, I. M., 1992. The matrix-degrading metalloproteinases. Bioassays 14:455–463.
505