Calcif Tissue Int (1992) 51:298-304

Calcified Tissue International 9 1992 Springer-Verlag New York Inc.

Interactions Between Growth Hormone and Dexamethasone in Skeletal Growth and Bone Structure of the Young Mouse A. Altman, 1 Z. Hochberg, 2 and M. Silbermann 1'3 Departments of 1Morphological Sciences and 2Pharmacology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 3The Rappaport Institute for Research in the Medical Sciences, P.O.B. 9649, Haifa, 31096 Israel Received January 7, 1992, and in revised form March 12, 1992

Summary. The present study investigated the interactions of growth hormone (GH) and glucocorticoid on skeletal growth and bone structure in young mice. The purpose of this study was to examine the possible prevention by GH of the damage inflicted by dexamethasone (Dex) at sites of skeletal growth and ossification. Dex (1 mg/kg) with or without rat GH (rGH) or bovine GH (bGH), 1 mg/kg, was given for 4 weeks, from age 3-7 weeks, to female ICR mice. Tibiae, humerus, and vertebrae were analyzed morphometrically and biochemically. Growth, as determined by the mouse weight, tibial length, and humerus protein content was found to be compromised by dexamethasone. This was prevented by rGH or bGH. The epiphyseal growth plate width, trabecular bone volume, cortical bone width, mineral bone content, and alkaline and acid phosphatase activity were decreased by dexamethasone. These were prevented by rGH or by bGH. The findings of the present study suggest that in the mouse, GH can decrease or even avoid some of the pathological features in growing bones inflicted by high-dose glucocorticoid treatment.

Materials and Methods Materials Rat GH (rGH) GH-B-12 (AFP-10478C) was provided by the Pituitary Hormones Distribution Program, NIDDK-Bethesda, MD. Recombinant Bovine GH (bGH) was a gift from BioTechnology General Ltd. (Rehovot, Israel). Dexamethasone sodium phosphate and sodium meta-bisulphite (Dexacort) are products of Teva (Jerusalem, Israel). P-nitro phenol phosphate (PNPP) and bovine serum albumin (BSA) are the product of Sigma Chemical Co. (St. Louis, MO).

Animals Locally bred ICR female mice weighing 15-16 g were taken at the age of 3 weeks. The animals were fed with standard pellet food containing 20% protein, 0.6% calcium, 0.5% phosphorus, and 0.2% magnesium. Food and water were given ad libitum. The animals were weighed and their length was measured three times per week. The protocol was approved by the ethical committee of the Faculty of Medicine, Technion Israel Institute of Technology.

Key words: Dexamethasone - Growth hormone - Bone mineral c o n t e n t - Glucocorticoids.

Experimental Protocol

Elevated concentration of glucocorticoids has been reported to cause skeletal growth retardation in growing animals and children [1], yet the secretion of growth hormone (GH) in these individuals was found to be normal [2, 3]. On the other hand, the levels of IGF-1 were found to be low [4, 7]. Glucocorticoids were reported to enhance bone resorption, both in vivo and in vitro [8], and to inhibit bone formation [9-11]. The latter takes place due to apparent changes in the synthesis or receptor binding of locally produced factors that regulate bone formation [12]. Skeletal ceils have been shown to synthesize insulin-like growth factor-1 (IGF-1), a known stimulator of bone cell replication and collagen synthesis [13]. Cortisol was reported to decrease skeletal IGF-1 synthesis [14]. Indeed, parathyroid hormone (PTH) and GH are among the major stimulators of IGF-I production by bone cells [13, 15]. The present study was undertaken to evaluate the eventual preventing effect of GH therapy on growth attenuation and bone loss during glucocorticoid administration in a model system of young mice.

Offprint requests to: M. Silbermann

Six groups of five mice each were injected s.c. five times per week for 4 weeks as follows: group 1 (control): saline 0.5 ml; group 2 (dexamethasone) (Dex): 1 mg/kg body weight; group 3 (rGH): 1 mg/kg body weight; group 4 (bGH): 1 mg/kg body weight; group 5 (Dex + rGH): Dex 1 mg/kg body weight and rGH 1 mg/kg body weight; group 6 (Dex + bGH): Dex 1 mg/kg body weight and bGH 1 mg/kg body weight. These treatment doses were chosen after a pilot dose-response experiment, and the minimal doses that produced maximal effects were used (data are not shown). The animals were killed after 4 weeks of treatment by an overdose of ether, 1 day after the last injection. Tibiae, humeri, and lumbar vertebrae were gently dissected for analysis. Organs from one side were snap frozen in liquid nitrogen and stored at -70~ Other tissues were fixed in 10% neutral-buffer formaldehyde for 48 hours and then decalcified by 10% EDTA solution in 4~ for 10-14 days. Proteins were determined by the Lowry procedure after an overnight hydrolysis in 0.5 N NaOH.

Histology and Histomorphometry. Serial sections of 5 ~m each were prepared and stained with hematoxylin and eosin (H&E). For histomorphometric studies we used a computed image analysis system (Olympus Corp.-CUE-2 Lake Success, NY) with the C-2 Morphometry Version (Galai Corp. Migdal-Haemek-Israel) [16]. The system is connected to a Zeiss Axiomat Photomicroscope which is connected to a video camera (Panasonic wv-cd50 Tokyo, Japan). The image is transformed directly from the microscope to an IBM-PC-AT computer and a color monitor by the video camera. The parameters that were measured were (1) trabecular bone vol-

A. A l t m a n et al.: G H and D e x a m e t h a s o n e in Skeletal Growth and Structure

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Fig. 2. The effect of Dex, b G H , and r G H on tibial length o f 3-week-old I C R female mice. M e a n + SEM, n = 5. *P < 0.01 v e r s u s control; **P < 0.01 v e r s u s Dex; ***P < 0.001 v e r s u s Dex.

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Fig. 3. The effect o f Dex, b G H , and r G H on the tibiae growth plate width. M e a n -+ SEM, n = 5. *P < 0.01 v e r s u s Dex; **P < 0.02 v e r s u s Dex; ***P < 0.10 v e r s u s control; ****P < 0.02 v e r s u s control.

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A. Altman et al.: GH and Dexamethasone in Skeletal Growth and Structure

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Fig. 4. The effect of Dex, bGH, and rGH on soluble and total protein of the humerus bone shaft in 7-week-old ICR female mice. *P < 0.01 versus control; **P < 0.001 versus Dex.

Fig. 5. The condylar region of tibiae epiphysis (x150, H&E). (A) Control-saline treatment. (B) Dex treatment--a massive bone loss (long arrows) and absent cementing line with expansion of the bone marrow (B) are demonstrated in the condylar trabeculae structure

(T). (C) rGH + Dex treatment--most of the condylar trabeculi are preserved. (D) bGH + Dex treatment--most of condylar trabeculi are preserved; there is a mild bone loss compared with the Dex treatment.

ume (TBV) as percent of the total bone volume, (2) cortical bone width, and (3) epiphyseal growth plate width.

measured. The bones were then ashed for 24 hours in a 530~ oven, and the ash was weighed.

Bone Ashing. A fragment of the spinal column containing the five

Chemistry. The dissected bones were weighed and homogenized. After an overnight hydrolysis with 0.5 N NaOH, the supernatants were analyzed for alkaline and acid phosphatase by standard analytical methods [18]. Enzyme units were defined as the amount of

lumbar vertebrae were gently dissected, the soft tissues were removed, and the wet weight was measured. After an overnight dehydration in ether:ethanol h l (vol:vol) solution, the dry weight was

A. Altman et al.: GH and Dexamethasone in Skeletal Growth and Structure

301

Fig. 6, Tibiae bone shafts, longitudinal sections (• 150, H&E). (A) Control-saline treatment. (B) Dex treatment--severe trabecular bone loss (arrows) of the secondary spongiosa; the "shadows" (empty space that was bone matrix before) indicate the areas of bone

loss. (C) rGH + Dex treatment--most of the trabeculae (T) in the secondary spongiosa are preserved. (D) bGH+Dex--secondary spongiosa region. It is clearly demonstrated that the trabecular bone loss is minimized.

enzyme required to release 1 mmol of phosphate from the substrate, over 1 second at 37~

served it (355.2 txm, P < 0.02 versus Dex) (Fig. 3). Bone protein determinations of humerus bone shaft revealed a significant decrease of protein concentrations in Dex-treated animals (30.8 mg/g bone) compared with controls (60.5 mg/g bone P < 0.001) (Fig. 4). Dex + GH preserved proteins levels to 70 Ixg/mg, and to 68 txg/mg for rGH and bGH, respectively (P < 0.001 versus Dex).

Statistics. Data were analyzed by the Student's t test for unpaired data. Two-tailed analyses of variance (ANOVA) were used to compare the six treatment groups. Results are presented as mean -+ SEM.

Results

Histology Growth Figure 1 presents the weight increment of the treated mice. The mean weight increments of the Dex-treated mice was 18.9 g/4 weeks c o m p a r e d with 27 g of control animals (ANOVA, P < 0.01). When b G H was added to Dex, the mean weight increment was 23.3 g (ANOVA, P < 0.05 versus Dex). The effect of r G H was not significant. The tibial bone length is shown in Figure 2. Dex-treated mice showed a significant shortening of the tibiae (l.37 cm) compared with control (1.73 cm P < 0.02) whereas those treated with the combinations Dex + G H (1.6 cm for rGH and 1.62 cm for bGH) were indistinguishable from the control and significantly longer than the Dex group (P < 0.01). Regarding the growth plate width of tibia, Dex inhibited it (295.4 txm versus control 354.15, P < 0.001) and GH pre-

Figure 5 shows characteristic a p p e a r a n c e of the tibial condyle region. The Dex-treated condyles show marked bone trabecular loss. Both bGH and rGH treatments prevented, to a large extent, the Dex damage. Under Dex treatment, the trabeculae are small in size, and shrunken; there are no cement lines to indicate bone remodeling. The bone marrow cells show typical effects of steroid, the cells are small, some of them pycnotic, and severe fatty change is observed throughout the bone marrow. Figure 6 presents the characteristic bone loss of the trabeculae of the secondary spongiosa region in the Dex-treated animals compared with controls. The combination of Dex and GH preserved most of the secondary spongiosa trabeculae intact. Histomorphometrical measurements are shown in Fig-

302

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A. Altman et al.: GH and Dexamethasone in Skeletal Growth and Structure

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Fig. 7. The effect of Dex, rGH, and bGH on the TBV of the tibiae, as percentage of the total bone volume. Mean +_ SEM, n = 5. *p < 0.01 versus Dex; **P < 0.001 versus Dex; ***P < 0.01 versus control.

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Fig. 8. The effect of Dex, rGH, and bGH on the cortical bone width of tibiae. Mean + SEM, n = 5. *P < 0.001 versus control.

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ures 7 and 8. The TBV as percentage of the total bone volume was markedly decreased by Dex (Dex 9.8%) to less than 50% of control (22.2%, P < 0.01). Animals that were treated with both Dex and GH maintained normal values of TBV (Fig. 7). No significant changes were found in the cortical bone width between the Dex group and the combined Dex + GHtreated mice, (Fig. 8), however, the Dex dose inhibited the cortical bone width in the presence of GH.

Chemistry A significant decrease in bone mineral content of five lumbar vertebrae was observed in Dex-treated mice (27 mg) compared with controls (40 mg P < 0.01) (Fig. 9). The Dex + GH groups preserved most of the bone mineral content (35 mg, P < 0.01). Alkaline p h o s p h a t a s e activity was significantly reduced in the Dex-treated animals (1.56) compared with controls (3.4 U/mg; P < 0.001) Table 1. The animals treated with D e x + G H p r e s e r v e d their enzyme activity. Acid phosphatase was found to be lower in the Dex-treated mice (0.5 U/g) compared with controls (0.8 U/g). The animals treated with both GH and Dex preserved their enzyme activity at

control levels. GH therapy by itself stimulated acid phosphatase activity.

Discussion

The present study was undertaken to examine the effects of GH on growth and bone formation and function adversely affected by pharmacological doses of glucocorticoids. Recent reports suggest that GH and glucocorticoids may regulate reciprocally fuel metabolism [18] and immune functions [19]. Previously reported deleterious effects of glucocorticoids were confirmed, and we found that GH therapy prevented this damage to a large extent. The model system of the present study was previously used in our laboratory, with documentation of bone damage by glucocorticoids both in vivo and in vitro [20-22]. We elected to use bovine GH, and its closer resemblance than hGH to mouse GH minimized its antigenicity. Indeed, measurements of antibodies showed anti-hGH antibodies (> 1% binding in undiluted serum) in 25% of mice within 6 days, and 100% within 2 weeks of hGH treatment (data not shown). Low titer anti-bGH antibodies were found after 4 weeks of b G H treatment in only 15% of the animals. Rat GH is even less antigenic. The results with rGH showed similar, or slightly weaker biological

A. Altman et al.: GH and Dexamethasone in Skeletal Growth and Structure

Table 1. Bone homogenate alkaline and acid phosphatase specific activity in tibiae of dexamethasone and GH-treated mice

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3.40 - 0.22 1.56 - 0.09 3.71 + 0.27 4.28 -+ 0.22 4.05 _+ 0.49 4.04 --- 0.21

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Fig. 9. The effect of Dex, rGH, and bGH on the absolute amounts of mineral content of five lumbar vertebrae. Mean -+ SEM, n = 5. *P < 0.001 versus control; **P < 0.01 versus Dex.

The results of this animal study encourage human clinical trials of hGH therapy in glucocorticoid-treated patients.

References

effects than did bGH. Both hormones are somatogenic in their receptor binding, and we have recently demonstrated that bGH binds to mouse GH-receptor and GH-binding protein with higher affinity than does rGH (unpublished data). The dose of Dex was calculated to give an equivalent human dose of 0.1 mg/kg. Due to the high surface area of 16-25 g mice, the above dose is multiplied by 10. Growth deceleration by Dex, as measured by body weight and tibial length, was preserved by GH therapy significantly, yet incompletely. On the other hand, the epiphyseal growth plate width shortening by Dex was completely prevented by GH therapy. The width of a growth plate is the sum of cartilage proliferation and enchondral ossification. GH influences every single step along this cascade of events, partly directly and partly through IGF-I mediation [23, 24]. Possible sites of Dex damage that would result in narrowing of the growth plate on the one hand, and improvement in GH therapy on the other hand, include decreased GH secretion [25-27] (although this is controversial [4]), decreased GH induction of local IGF-I synthesis [28, 29], or increased circulating IGF-I inhibitors [7]. The preservation of normal IGF-I levels under Dex treatment (data not shown) does not exclude any of these mechanisms. Ossification-resorption damage by dexamethasone, as documented by decreased trabecular bone volume and cortical width of the tibiae and by decreased protein concentrations in the humerus, were completely prevented by GH therapy. The method e m p l o y e d does permit us to conclude whether it was due to cells or matrix depletion. We have also not measured the amount of fat that replaced some of the tissue. Similarly, alkaline phosphatase as a measure of osteoblastic activity, and acid phosphatase as a measure of osteoclastic activity were completely preserved by therapy. On the other hand, bone mineral content loss by Dex was incompletely, yet significantly prevented by GH. The welldocumented osteopenia after glucocorticoid treatment has been attributed to increased bone resorption and decreased bone formation [30-32[. These effects are partly direct, through inhibition by glucocorticoids of RNA generation and protein synthesis, as part of their catabolic effects, and through direct inhibitory effect on cell replication in bone [33-35]. Glucocorticoid effects are partly induced by PTH increase [12, 36] both directly and through inhibition of intestinal calcium absorption [37]. GH and IGF-I have clear anabolic and proliferative effects in a variety of tissues and specific stimulatory effects of bone formation [38, 39].

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A. Altman et al.: GH and Dexamethasone in Skeletal Growth and Structure

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