Reversible skeletal abnormalities in gamma-glutamyl transpeptidase-deficient mice

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Endocrinology 144(7):2761–2764 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-0071

Reversible Skeletal Abnormalities in ␥-Glutamyl Transpeptidase-Deficient Mice REGIS LEVASSEUR, ROBERTO BARRIOS, FLORENT ELEFTERIOU, DONALD A. GLASS, II, MICHAEL W. LIEBERMAN, AND GERARD KARSENTY Departments of Molecular and Human Genetics (R.L., F.E., D.G., G.K.) and Pathology (R.B., M.W.L.), M.D./Ph.D. Program (D.G.), Baylor College of Medicine, Houston, Texas 77030 ␥-Glutamyl transpeptidase (GGT) is a widely distributed ectopeptidase responsible for the degradation of glutathione in the ␥-glutamyl cycle. This cycle is implicated in the metabolism of cysteine, and absence of GGT causes a severe intracellular decrease in this amino acid. GGT-deficient (GGTⴚ/ⴚ) mice have multiple metabolic abnormalities and are dwarf. We show here that this latter phenotype is due to a decreased of the growth plate cartilage total height resulting from a proliferative defect of chondrocytes. In addition, analysis of vertebrae and tibiae of GGTⴚ/ⴚ mice revealed a severe osteopenia. Histomorphometric studies showed that this low bone mass phenotype results from an increased osteoclast number and activity as well as from a marked decrease in osteoblast activity. Interestingly, neither osteoblasts, osteoclasts, nor chondrocytes express GGT, suggesting that the

observed defects are secondary to other abnormalities. Nacetylcysteine supplementation has been shown to reverse the metabolic abnormalities of the GGTⴚ/ⴚ mice and in particular to restore the level of IGF-1 and sex steroids in these mice. Consistent with these previous observations, N-acetylcysteine treatment of GGTⴚ/ⴚ mice ameliorates their skeletal abnormalities by normalizing chondrocytes proliferation and osteoblastic function. In contrast, resorbtion parameters are only partially normalized in GGTⴚ/ⴚ N-acetylcysteinetreated mice, suggesting that GGT regulates osteoclast biology at least partly independently of these hormones. These results establish the importance of cysteine metabolism for the regulation of bone remodeling and longitudinal growth. (Endocrinology 144: 2761–2764, 2003)

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were killed was used to determine active mineralization sites and rates of bone formation (4).

GT (␥-GLUTAMYL TRANSPEPTIDASE) is a widely expressed ectopeptidase responsible for the degradation of glutathione that plays an important role in cysteine metabolism (1). Mice deficient in GGT (GGT⫺/⫺ mice) are dwarf and present multiple endocrine defects (2, 3). These endocrine abnormalities can be rescued by treatment with N-acetylcysteine (NAC) (2, 3). The short stature of GGT⫺/⫺ mice led us to study their growth plate (GP). Histological analyses of the GP cartilage of 6-wk-old GGT⫺/⫺ mice revealed a decrease in chondrocyte proliferation. Our analysis also uncovered that GGT deficiency causes an osteopenia due to both decreased bone formation and increased bone resorption. Upon rescue of the hormonal defect by NAC supplementation cartilage and bone formation, defects are fully rescued, whereas bone resorption parameters remain partly abnormal. These results establish that GGT indirectly regulates multiple aspects of skeletal biology. Materials and Methods

GP morphometry and bromodeoxyuridine (BrdU) labeling Tibiae were decalcified and embedded in paraffin. Seven-micrometer sections were stained with hematoxylin and eosin. Morphometry of the GP was performed using a camera with Spot RT software (Diagnostic Instruments, Sterling Heights, MI) on longitudinal sections, close to the midline of the bone. The total height of the GP (TH) was defined as the distance between the two chondrosseous junctions (5). The heights of the proliferative and hypertrophic zone (PZ and HZ) were measured based on morphological criteria (6). The height of the resting zone (RZ) was defined as RZ ⫽ TH – (PZ ⫹ HZ) (5). For BrdU labeling, 6-wk-old mice were injected (ip) with 100 ␮l of 100 ␮m BrdU (Sigma), killed 1 h later, and tibiae were processed in paraffin. Sections were stained using a BrdU staining kit (Zymed Laboratories, Inc., South San Francisco, CA). Cells were counted in the same area in all sections and expressed as the percentage of the BrdU-positive cells vs. the total number of cells.

X-ray analysis and bone histomorphometry

Animals studies The generation of GGT⫺/⫺ mice has been previously reported (2). GGT⫺/⫺ and wild-type (WT) mice were maintained on C57BL6/ 129SvEv genetic background. For rescue experiments, NAC (Sigma, St. Louis, MO) was dissolved in the drinking water (10 g/liter) and supplied ad libitum to 3-wk-old GGT⫺/⫺ mice until 6 wk of age. Calcein labeling (15 mg/kg, ip; Sigma) at 10 d and 2 d before the mice Abbreviations: BFR, Bone formation rate; BrdU, bromodeoxyuridine; CT, computerized tomography; DPD, deoxypyridinoline; GGT, ␥glutamyl transpeptidase; GP, growth plate; HZ, hypertrophic zone; NAC, N-acetylcysteine; PZ, proliferative zone; RZ, resting zone; TH, total height of the GP; WT, wild-type.

X-ray analysis was performed using a Faxitron (Philips, Wheeling, IL). Histology was performed on female mice. Vertebrae and tibiae were dissected and embedded in methyl methacrylate. Seven-micrometerthick frontal (vertebrae) and sagittal sections (tibiae) were stained according to the Von Kossa method (7). One 7-␮m-thick section per animal was stained for tartrate-resistant alkaline phosphatase activity and counterstained with toluidine blue for cell counting. One 12-␮m-thick section per animal was left unstained for dynamic measurements of bone formation. Histomorphometric data were collected with the Osteomeasure system and the Trabecular Analysis System (Osteometrics, Atlanta, GA). Data are reported in accordance with standard nomenclature (8). Micro-CT (computerized tomography) analysis was performed on Scamco NCT40 (Scamco, Bassersdorf, Switzerland).

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Immunohistochemistry and histochemistry Tissue sections were blocked with H2O2 in methanol. Antigen retrieval was performed using citrate buffer. A primary rabbit polyclonal antibody against ␥-GTT developed in our laboratory (1/100) and a secondary biotinylated anti-Ig followed by horseradish peroxidaseconjugated streptavidin (BioGenex Laboratories, Inc., San Ramon, CA) were used. 3-Amino-9-ethyl-carbazole (BioGenex Laboratories, Inc.) in acetate buffer containing 0.05% H2O2 was used as substrate. The sections were counterstained with hematoxylin.

RIA and statistical analysis Deoxypyridinoline (DPD) cross-links were measured in morning urines using the Pyrilin KS-D immunoassay kit (Metra Biosystems, Mountain View, CA). Creatinine values were used for standardization between samples (creatinine kit; Metra Biosystems). Estradiol and IGF-I serum levels were measured by RIA and EIA (Diagnostic Systems Laboratories, Webster, TX), respectively. Data are expressed as the mean ⫾ se. Statistical analysis was performed by one-way ANOVA. Values were considered statistically significant at P ⬍ 0.05.

Results and Discussion Growth plate abnormalities in GGT⫺/⫺mice

Levasseur et al. • Brief Communications

less of their sex (6.2 ⫾ 0.1 mm in GGT⫺/⫺ males vs. 11.6 ⫾ 0.2 mm in WT males, P ⬍ 0.001) (Fig. 1). Analysis of the GP cartilage of the GGT⫺/⫺ mice revealed that its total height was reduced by more than 20% compared with WT mice (Fig. 2 and Table 1). Accordingly, the height of the proliferative and of the hypertrophic zone was decreased in GGT⫺/⫺ mice (Table 1). We also observed a decreased number of chondrocytes in the proliferating and hypertrophic zone (Table 1). In contrast, the height of the resting zone was increased in the mutant mice. To determine whether these cell abnormalities were due to a decrease in chondrocyte proliferation, we performed BrdU labeling in vivo. As expected, the percentage of BrdU-positive cells per total number of cell was markedly decreased in the proliferating zone of GGT⫺/⫺ mice compared with WT littermates (Table 1). We also measured the mean height of hypertrophic chondrocytes and found it to be decreased in GGT⫺/⫺ mice (Table 1). Thus, the decreased total height of the GP results both from decreased proliferation of chondrocytes and from an atrophy of hypertrophic chondrocytes.

GGT⫺/⫺ mice become dwarf soon after birth (2). At 6 wk of age, their tibia length was reduced by nearly 30% regard-

Osteopenia in GGT⫺/⫺ mice

FIG. 1. X-ray analysis of 6-wk-old male WT, GGT⫺/⫺, and NACtreated GGT⫺/⫺ mice. Note the dwarfism and the decreased bone density in GGT⫺/⫺ mice compared with WT and the rescue of dwarfism and bone density in GGT⫺/⫺ NAC-treated mice.

In addition to a reduction of every skeletal element length, x-ray revealed a marked decrease in bone density in GGT⫺/⫺ mice, regardless of their sex (Fig. 1 and data not shown). At 6 wk of age, long bones and vertebrae of mutant mice appear more lucent than their WT littermate. Consistent with this observation, histological analysis showed that bone volume was decreased 30 – 40% in GGT⫺/⫺ mice compared with WT (Fig. 3). Cortical thickness measured by micro-CT was decreased in GGT⫺/⫺ mice (0.145 ⫾ 0.002 mm vs. 0.166 ⫾ 0.009 mm in WT mice, P ⬍ 0.002). We next assessed the functional bases of this phenotype. Osteoclast number, osteoclast surface, and eroded surface were increased in GGT⫺/⫺ mice, (Table 2) as well as the urinary elimination of DPD cross-links, a biomarker of bone resorption (16.5 ⫾ 1.4 nmol DPD/mmol creatinine in WT vs. 36.4 ⫾ 3.0 nmol DPD/mmol creatinine in GGT⫺/⫺ mice, P ⬍ 0.001). There was also a significant decrease of mineral apposition rate and of the bone formation rate (BFR) in

FIG. 2. GP analysis of the tibia of 6-wk-old male WT, GGT⫺/⫺, and NAC-treated GGT⫺/⫺ mice. Note the decrease of the total height (double arrow) of the GP in GGT⫺/⫺ mice and the rescue by NAC.

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GGT⫺/⫺ mice compared with WT mice (Table 2). The number of osteoblasts was normal, suggesting that osteoblast function rather than osteoblast proliferation was affected. Consistent with these observations, trabecular thickness and number were reduced, whereas trabecular space was increased in GGT⫺/⫺ mice (Table 2). Taken together, these results indicate that the osteopenic phenotype of the GGT⫺/⫺ mice is caused by both increased bone resorption and decreased bone formation. Absence of expression or activity of GGT in bone

To assess whether the skeletal phenotype observed in the GGT-deficient mice was cell autonomous, we analyzed GGT expression in bone. Immunohistochemistry studies performed in tibiae did not detect the presence of GGT in the primary or secondary spongiosa nor in any chondrocytes of the GP cartilage in 6-wk-old WT male or female mice, whereas GGT immunoreactivity was readily detectable in kidney (Fig. 4 and data not shown). This result indicates that the skeletal abnormalities observed in absence of GGT are not cell autonomous.

Partial rescue of the skeletal abnormalities of GTG⫺/⫺ mice by NAC

Because GGT is not expressed in osteoblasts, osteoclasts, or chondrocytes, and GGT⫺/⫺ mice present abnormalities in serum levels of hormones known to influence skeletal biology, we tested whether restoring normal endocrine function would correct the skeletal growth and bone remodeling defects. NAC supplementation can rescue many phenotypic abnormalities in GGT⫺/⫺ mice, in particular restore normal levels of IGF-1 and sex steroids in serum (2, 3). We therefore treated GGT⫺/⫺ mice with NAC and analyzed their skeleton. NAC supplementation rescued the dwarfism of the GGT⫺/⫺ mice (Fig. 1). Tibia length was increased 26% compared with untreated GGT⫺/⫺ mice (14.7 ⫾ 0.1 mm vs. 11.6 ⫾ 0.2 mm, P ⬍ 0.001) and the total height of the GP became normal (Fig. 2 and Table 1). Accordingly, the height of the proliferative and hypertrophic zones became normal in NAC-treated GGT⫺/⫺ mice as was the mean size of the last hypertrophic chondrocytes (Table 1). Chondrocyte numbers in proliferative and hypertrophic zone was close to what is observed in WT GP, and BrdU labeling confirmed that TABLE 2. Morphometric data of vertebrae in 6-wk-old males WT

TABLE 1. Morphometric data of GP in 6-wk-old males

Total height (␮m) RZ height (␮m) PZ height (␮m) PZ cell number (column) HZ height (␮m) HZ cell number (column) HZ last cell (␮m) BrdU-positive cells (%)

WT

GGT⫺/⫺

GGT⫺/⫺ ⫹NAC

191.3 ⫾ 11.3 7.2 88.2 ⫾ 5.3 17.5 ⫾ 1.0

151.4 ⫾ 9.9a 30.0 58.6 ⫾ 5.2a 12.6 ⫾ 0.5a

180.4 ⫾ 13.4 10.2 83.2 ⫾ 6.2 16.1 ⫾ 1.1

95.9 ⫾ 6.2 5.5 ⫾ 0.2

a

62.8 ⫾ 5.8 4.9 ⫾ 0.2a

87.0 ⫾ 7.5 5.3 ⫾ 0.2

34.1 ⫾ 0.3 13.3 ⫾ 0.8

29.8 ⫾ 0.4a 8.7 ⫾ 0.8a

33.4 ⫾ 0.4 11.7 ⫾ 0.9

Values are the mean ⫾ SE. a Different from WT, P ⬍ 0.05.

Oc.Nb (/mm) Oc.S/BS (%) ES/BS (%) Ob.Nb (/mm) BFR (␮m3/ ␮m2/yr) MAR (␮m/d) Tb.Th (␮m) Tb.Nb (/mm) Tb.Sp (␮m)

GGT⫺/⫺

GGT⫺/⫺ ⫹NAC

3.5 ⫾ 0.8 9.2 ⫾ 0.7 15.0 ⫾ 0.9 14.8 ⫾ 2.0 304.6 ⫾ 43

7.3 ⫾ 1.3a 17.9 ⫾ 1.9a 25.4 ⫾ 1.3a 14.2 ⫾ 2.8 138.7 ⫾ 21a

5.1 ⫾ 1.0a 11.8 ⫾ 1.0a 19.1 ⫾ 1.0a 15.6 ⫾ 1.7 283.5 ⫾ 27

1.74 ⫾ 0.2 22.7 ⫾ 0.9 7.2 ⫾ 0.3 117.2 ⫾ 6.8

0.80 ⫾ 0.1a 16.9 ⫾ 0.3a 6.1 ⫾ 0.3a 151.5 ⫾ 11.7a

1.65 ⫾ 0.1 19.7 ⫾ 0.4a 7.0 ⫾ 0.3 123.2 ⫾ 6.6

Oc.Nb, Osteoclast number; Oc.S/BS, osteoclast surface per bone surface; ES/BS, eroded surface per bone surface; Ob.Nb, osteoblast number; MAR, mineral apposition rate; Tb.Th, trabecular thickness; Tb.Nb, trabecular number; Tb.Sp, trabecular space. a Different from WT, P ⬍ 0.05.

FIG. 3. Analysis of vertebrae of 6-wk-old male WT, GGT⫺/⫺ mice, and NAC-treated GGT⫺/⫺ mice. Note the decrease in bone volume (BV/TV, %) in GGT⫺/⫺ mice and the partial rescue of this phenotype in NAC-treated GGT⫺/⫺ mice. *, Different from WT, P ⬍ 0.05.

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FIG. 4. GGT immunoreactivity in bone (left panel) and kidney (right panel). Note the absence of GGT expression in bone but its presence in kidney.

NAC treatment restored a normal rate of chondrocyte proliferation (Table 1). Bone density as judged by x-ray appeared improved in NAC-treated GGT⫺/⫺ mice (Fig. 1), although bone volume (Fig. 3) and cortical thickness were not fully normalized (0.154 ⫾ 0.03 mm in GGT⫺/⫺ NAC mice vs. 0.166 ⫾ 0.0009 mm in WT). Osteoclast number and indices of osteoclast activity decreased but remained elevated in NACtreated GGT⫺/⫺ mice compared with WT mice (Table 2 and 28.94 ⫾ 6.5 nmol DPD/mmol creatinine in GGT⫺/⫺ NAC mice vs. 16.5 ⫾ 1.4 nmol DPD/mmol creatinine in WT). In contrast, mineral apposition rate and BFR were fully normalized in NAC-treated GGT⫺/⫺ mice (Table 2). As a consequence, trabecular number and space were normalized in GGT⫺/⫺ mice (Table 2), whereas trabecular thickness remained decreased. Moreover, NAC treatment normalized estradiol levels (6.18 ⫾ 0.08 pg/ml in NAC-treated GGT⫺/⫺ mice vs. 5.28 ⫾ 0.10 pg/ml in WT mice). It also largely corrected without normalizing the decrease in IGF-1 serum levels of GGT⫺/⫺ mice (3) (372.8 ⫾ 16.7 ng/ml in NACtreated GGT⫺/⫺ mice vs. 453.6 ⫾ 33.0 ng/ml in WT). Our study provides in vivo evidence for the critical influence cysteine metabolism has on skeletal growth and bone remodeling. It shows that the absence of GGT results in chondrocyte, osteoblast, and osteoclast abnormalities. As GGT is not expressed in these cells, it is likely that the decreased osteoblast and chondrocyte functions are the result of several interdependent metabolic and trophic changes present in GGT⫺/⫺ mice. The phenotype observed in these mice is part of an overall reduction in organ size (2), some of which is attributable to a reduction in circulating and intracellular cysteine levels (2, 9). The dwarfism in GGT⫺/⫺ mice could also be explained by the lack of IGF-I, as serum IGF-I regulates bone growth and density (10). We hypothesize that the low BFR is the result of lack of cysteine and/or IGF-I. The hypogonadism of the GGT⫺/⫺ mice may, however, not be solely responsible of their increased bone resorption because its correction by NAC treatment normalizes estradiol levels but does not fully normalize osteoclast number and activity. This observation suggests that, although many of the skeletal abnormalities observed in GGT⫺/⫺ mice are likely caused by their lack of IGF-I and steroid hormone, cysteine deficiency may cause other deleterious consequences resulting in osteopenia.

Acknowledgments We are grateful to Ms. D. Atwood for help with animal husbandry, to Dr. Kapadia (Scamco) for micro-CT measurements, and to L. Green for technical help. Received November 22, 2002. Accepted March 11, 2003. Address all correspondence and requests for reprints to: Gerard Karsenty, M.D., Ph.D., Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: [email protected] This work was supported by the National Space Biomedical Research Institute Grant NCC9-59, the National Aeronautics and Space Administration Grant MOD 1-FY-00-686, NIH Grant R01-DK58883 (to G.K.), NIH Grant R01-ES07827 (to M.W.L.), the Rheumatology French Society and the Philippe Foundation (to R.L.), the Children’s Nutrition Research Center (to F.E.), and the Bone Disease Bone Program of Texas.

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