Growth hormone inhibits differentiation of avian epiphyseal growth-plate chondrocytes

w

MOlecular and Cellular

Endocrinology

ELSEVIER

Molecular and Cellular Endocrinology 114 (1995) 35-42

Growth hormone inhibits differentiation of avian epiphyseal growth-plate chondrocytes Efrat Monsonegoa, Orna Halevyb, Arieh Gertler ‘, Shmuel Hurwitz”, Mark Pines*a aInstitute ofAnimal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, USA bDepartment of Animal Science, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel ‘Department of Biochemktry and Human Num.tion, The Hebrew Uniwrsi@ of Jerusalem, Faculty ofAgriculture, Rehovot 76100, Israel

Received 10 May 1995; accepted 17 July 1995

Abstract The effect of chicken growth hormone (cGH) on the proliferation and differentiation of avian growth-plate chondrocyte was evaluated in culture. In culture, addition of ascorbic acid to the culture media caused cell differentiation. Treatment of proliferating chondrocytes with cGH caused a time-dependent increase in collagen type II gene expression together with a decrease in the appearance of osteopontin (OPN) in the medium. In addition, the ascorbic acid-dependent increase in alkaline phosphatase (AP) activity was inhibited by cGH. IGF-I, on the other hand, caused an increase in AP activity in the ascorbic acid-treated chondrocytes. In the presence of ascorbic acid, cGH did not affect collagen type II gene expression or the appearance of OPN in the medium. Proliferation of avian growth-plate chondrocytes, in contrast to mammalian chondrocytes, was not stimulated by GH alone, although the presence of cGH was essential for chondrocyte survival in long-term culture. cGH in combination with epidermal growth factor (EGF) stimulated cell proliferation. These results suggest that GH inhibits differentiation in avian growth-plate chondrocytes, thereby sustaining their proliferative state and maintaining their sensitivity to growth factors such as EGF. Keywords: Alkaline phosphatase;

Collagen II; Osteopontin;

1. Introduction

Growth hormone (GH) plays a major role in longitudinal bone growth of mammalian species. Local administration of GH to the tibia1 epiphyseal growthplate of hypophysectomized rats stimulated unilateral bone growth, suggesting that GH acted directly on chondrocytes of the epiphyseal growth-plate (Isaksson et al., 1982; Isgaard et al., 1986; Schlechter et al., 1986; Nilsson et al., 1987). This action is apparently mediated by a single high affinity, membrane-associated receptor (GH-R) (Nilsson et al., 1986, 1989; Maor et al., 1989; Bentham et al., 1993). Furthermore, GH induces cell division in the germinal cell layer in

*Corresponding

author, Tel.: 972 8 470583; Fax: 972 8 475075.

Ascorbic acid; IGF-I

vivo (Ohlsson et al., 1992a) and stimulates chondrocyte colony formation in suspension cultures (Lindahl et al., 1986), which appears to be dependent on the stage of differentiation of the cells (Ohlsson et al., 1993). Some of the effects of GH on longitudinal bone growth are thought to be mediated through release of insulin-like growth factor I (IGF-I) from the liver into circulation (Daughaday, 1989) or locally by the chondrocytes (Isaksson et al., 19871, whereas in some cases direct and independent effects of GH could be demonstrated (Ohlsson et al., 1992a). The function of GH in regulation of avian growth in general and in longitudinal bone growth in particular, is not clear. Genetic strains of birds with the slowest growth rates exhibited the highest concentration of circulating GH (Goddard et al., 1988). Moreover, the average peak, frequency and amplitude of

0303-7207/95/%09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved, SSDI 0303-7207(95)03639-O

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E. Monsonego et al. /Molecular

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the spontaneous oscillation in plasma GH were higher in slow than in fast growing strains (Bacon et al., 1989). Administration of GH to young chickens did not alter growth performance (Scanes et al., 1984; Burke et al., 1987; Cogburn et al., 1989), or longitudinal bone growth and growth-plate width (Burke et al., 1987; Cravener et al., 1989). However, effects on carcass composition and an increase in body weight were observed in some cases (Scanes et al., 1990; Rosebrough et al., 1991). In culture, no effect of GH was observed on morphology, proliferation or metabolic activity of chondrocytes derived from avian growth-plate (Rosselot et al., 1992, 1994). Chondrocytes located in different regions of the growth-plate differ in their differentiation state, morphology, secretion of extracellular matrix components and activities of various enzymes. Synthesis of collagen type II characterizes chondrocytes at the proliferative state (Hinek et al., 1987; Oshima et al., 19891, whereas alkaline phosphatase (AP) activity and collagen type X synthesis are restricted to hypertrophic cells (Vaananen, 1980; Poole and Pidoux, 1989). Chondrocyte differentiation is accompanied also by the increased synthesis of a phosphorylated extracellular matrix protein - osteopontin (OPN) - which is believed to play an important role in the process of cartilage and bone mineralization (McKee, et al., 1992; Barak-Shalom et al., 1995; Knopov et al., 1995). In culture, chondrocytes appear to undergo differentiation which is augmented by ascorbic acid (Leboy et al., 1989; Halevy et al., 1994; Barak-Shalom et al., 1995). Using cell cultures, we demonstrated recently that avian growth plate chondrocytes expressed the gene coding for the GH-R regardless of the stage of cell differentiation (Monsonego et al., 1993). In the present study, we demonstrate inhibition of chondrocyte differentiation in culture by chicken growth hormone (cGH), by examining its effects on collagen type II, OPN synthesis and AP activity. 2. Materials and methods 2.1. Materials [methyl-3H]Thymidine (93 Ci/mmol) and ATP (a32P 6000 Ci/mmol) were obtained from The Radiochemical Centre (Amersham, England). Dulbecco’s modified Eagle’s medium (DMEM), trypsin-EDTA solution (0.25-0.02%) and basic fibroblast growth factor (bFGF) were obtained from Sigma (St. Louis, MO, USA). Fetal calf serum (FCS) and serum-free medium-Bio-MPM-1 developed for anchorage-dependent cells were obtained from Biochemical Industries (Beth-Haemek, Israel). Recombinant cGH was a gift from Bio-Technology General Ltd. (Rehovot, Israel). Epidermal growth factor (EGF) and goat anti-rabbit

Endocrinology 114 (1995) 35-42

IgG conjugated to peroxidase were from BioMakor (Rehovot, Israel). Recombinant IGF-I was obtained from Genzyme (Boston, MA, USA). The cDNA probe for the chicken collagen type II-ORl-derived from the 890 bp of type II collagen genomic DNA fragment (BamHI/PstI), was prepared as previously described (Granot et al., 1993). Chicken OPN probe and OPN antiserum were prepared as described elsewhere (Barak-Shalom et al., 1995). 2.2. Cell culture Avian epiphyseal growth-plate chondrocytes were prepared and cultured as described previously (Pines and Hurwitz, 1988). Only early passages (one to three) were used. Prior to the experiments, the cells were detached from the plastic dish by incubation with trypsin-EDTA solution, plated in DMEM containing 5% FCS and allowed to resume development until reaching confluency. For differentiation studies, cells were cultured in DMEM containing 5% FCS with daily addition of ascorbic acid. For long-term serumfree cultures, Bio-MPM-1 medium was used. 2.3. Cell proliferation After 20 h incubation with the appropriate medium, fresh medium containing 1 pCi/ml [3H]thymidine was added to the culture for an additional 4 h. At the end of incubation, the media were discarded and the cells were detached from the dish by incubation with trypsin-EDTA solution and treated with 4% perchloric acid. The resulting precipitate was washed and the DNA-bound [3H]thymidine was estimated as described previously (Pines et al., 1988). For the longterm experiments, direct estimations of cell number were made using a cell counter (Coulter Electronics, Luton, England). 2.4. RNA isolation and Northern blot analysis Total cell RNA was extracted by the acid guanidium-phenol-chloroform method as described previously (Granot et al., 1991). The samples (10 or 20 pg) were subjected to electrophoresis through a 1% agarose gel containing 6% formaldehyde, after which the RNA was blotted onto a Nytran membrane (New England Nuclear, Boston, MA, USA). Hybridization with cDNA probes for collagen type II, OPN and p-actin was carried out at 42°C in buffer containing 50% formamide. The filters were washed and autoradiographed. 2.5. Immunoblotting After incubation, the media were collected, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% SDS-PAGE) and transferred to nitrocellulose (Pines et al., 1985). The membranes were blocked for 2 h at 25°C with 5% dried skimmed

E. Monsonego et al. /Molecular and Cellular Endocrinology 114 (1995) 35-42

milk in Tris-buffered saline (TBS; 20 mM Tris/HC1/500 mM NaCl, pH 7.5). The OPN antiserum was used at a 1:500 dilution in 1% dried skimmed milk/TBS for 2 h. The blots were washed extensively with 0.1% Tween 2O/TBS and incubated with goat anti-rabbit IgG coupled to horseradish peroxidase (1:lOOOO dilution) in 1% dried skimmed milk/TBS for 1 h. The blots were washed as above before using the enhanced chemiluminescence detection system (Amersham, England).

37

dose-dependent decrease in collagen type II gene expression concomitant with an increase in OPN gene expression and AP activity (Fig. 1). AP activity was already maximal at 1 pg/ml ascorbic acid, whereas 10 pg/ml was needed for maximal changes in type II collagen and OPN gene expression. 3.2. Effect of cGH on collagen II gene expression The effect of cGH on type II collagen gene expression was studied in ascorbic acid-treated and nontreated chondrocytes (Fig. 2). In the non-treated cells, cGH (100 ng/ml) augmented type II collagen gene expression which was time-dependent with maximum expression after 24 h (Fig. 2A). No alteration in collagen type II gene expression in response to cGH (100 ng/ml) was observed in chondrocytes treated for 3 days with ascorbic acid (Fig. 2B).

2.6. Alkaline phosphatase activity assay The cell layer was washed twice with saline, after which the cells were scraped and collected into assay buffer (0.1 M glycine, 1 mM MgCl,, 85 mM NaOH, pH 10.5). An aliquot (100 ~1) of the solubilized cell extract was taken for assay. Enzyme activity was measured at 410 nm after incubation with 5 mM pnitrophenol phosphate (Sigma 104) as substrate in the assay buffer. AP activity was expressed as units of p-nitrophenol formed/min/mg protein.

3.3. Effect of cGH on alkaline phosphatase activity In the absence of ascorbic acid, low AP activity was observed which was not affected by cGH at all concentrations tested (up to 200 ng/ml). Treatment with ascorbic acid for 48 h (1 pug/ml) caused a marked increase in AP activity (Fig. 3). Under those conditions, chicken GH prevented the increase in enzyme activity in a dose-dependent manner, and at a concentration of 200 ng/ml AP activity was reduced to near basal level (Fig. 3A). After 96 h of ascorbic acid treatment, the cells no longer responded to cGH by changes in AP activity (data not shown). In a different set of experiments IGF-I increased AP activity of the ascorbic acid-treated chondrocytes without any sig-

3. Results 3.1. Effect of ascorbic acid on chondrocytes differentiation

When cultured in the presence of serum, avian growth-plate chondrocytes expressed high levels of collagen type II mRNA, low levels of OPN mRNA (Fig. 1A) and exhibited low AP activity (Fig. lB), suggesting that the cells were in their proliferative state. Incubation with ascorbic acid for 48 h caused a A Collagen

ccl(Il)

Osteopontin

0 Ascorbic

0.1

mRNA

Alkaline

phosphatase

activity

mRNA

I

10

acid (pglml)

I--\\ 0

. .. .(

.

. ..___, . . 1

.I

Ascorbic

acid

.._I0

(jig/ml)

Fig. 1. Effect of ascorbic acid on the expression of the genes coding for collagen type II and OPN and on AP activity. Cells were incubated in DMEM containing 5% FCS with various concentrations of ascorbic acid for 48 h. At the end of the incubation period, total RNA was extracted and subjected to electrophoresis and blotting. The same blot was hybridized with chicken-specific collagen type II and OPN (A) 32P-labeled probes. Each lane was loaded with 10 pg RNA. The amount of RNA loaded per lane was monitored by methylene blue staining of 18s and 28s RNA. Parallel plates were used for evaluation of AP activity (B). The results are the mean f SE of three experiments.

38

E. Monsonego et al. /Molecular and Cellular Endocrinology 114 (1995) 35-42

A

,,“(_>

0

Collagen al(II) p Actin

Time (h)

1

0

5

CCH(loo

19

24

n&w) 0

10

20

200

cGH (nglml)

B Collagen al(II) cGH (lOOn@d) Ascorbic

acid

(lpghnl)

_

+

-

+

-

-

+

+

100

I(;F-I Fig. 2. Effect of cGH on collagen type II gene expression. (A) Time-dependence effect of cGH on collagen type II gene expression. Cells were incubated with cGH (100 ng/ml) in ascorbic acid-free media for the time indicated. Total RNA was extracted and each lane was loaded with 20 pg. After electrophoresis and blotting, the RNA was hybridized with 32P-labeled probes for chicken collagen type II (ORl) or p-actin. (B) Cells were cultured with or without ascorbic acid (1 pg/ml/day) for 3 days, after which the media were changed to fresh media with or without cGH (100 ng/ml) for an additional 24 h. Total RNA was extracted and each lane was loaded with 10 pg. After electrophoresis and blotting, the RNA was hybridized with 32P-labeled probe for chicken collagen type II (OR0 The amount of RNA loaded per lane was monitored by methylene blue staining of 18s and 28s RNA.

nificant effect on chondrocytes not treated with ascorbic acid (Fig. 3B). 3.4. Effect of cGH on the appearance of OPN in the culture medium Chondrocytes treated for 3 days with ascorbic acid, secreted elevated levels of OPN into the medium compared with non-treated cells (Fig. 4, lanes 1 and 3). Incubation with cGH resulted in a decrease in the OPN levels in the media only of the non-treated cells (Fig. 4, lane 21, whereas in the ascorbic acid-treated cells no changes in OPN levels by cGH were observed (Fig. 4, lanes 2 and 4). 3.5. GH and chondrocyte proliferation Replacement of 5% FCS-containing DMEM with serum-free Bio-MPM-1 medium resulted in a decrease in chondrocyte number, and after 4 days very

(nglmlt

Fig. 3. Effect of cGH (A) and IGF I (B) on ascorbic acid-dependent AP activity. Cells were incubated with various concentrations of cGH (A) or IGF I (B) with or without 1 pg/rnl ascorbic acid for 48 h. At the end of the incubation period, AP activity was determined at 410 nm using p-nitrophenol phosphate as substrate. The results are the mean f SE of three experiments.

few cells survived. However, daily addition of cGH (100 ng/ml) abolished the observed decrease in cell number. Incubation of the cells in the presence of IGF-I (100 ng/ml) increased cell number (Fig. 5). Chondrocytes incubated with human or chicken GH together with EGF (at a concentration which by itself causes only minor increase in cell proliferation) exhibited an increase of 1.7- and 2.3-fold in [3H]thymidine incorporation, respectively, compared with cells incubated with either peptide alone (Fig. 6). The homologous chicken hormone appeared more potent than the heterologous one (Fig. 6), although it binds with lower aftinity to the chicken GH receptor (Monsonego et al., 1993). An increase in cell proliferation was also observed when cGH was incubated together with bFGF or IGF-I (at concentrations of 1 and 10 ng/ml, respectivly), but to a lesser extent than with EGF (data not shown). 4. Discussion

Avian growth-plate chondrocytes in culture are ideally suited for studying the effect of various hormones and growth factors on proliferation, matrix protein

39

E. Monsonego et al. /Molecular and Cellular Endocrinology 114 (1995) 35-42

Osteopontin

Ascorbic

acid

-

-

+

-f=

Fig. 4. Effect of cGH on OPN appearance in the medium. Cells were cultured with or without ascorbic acid (1 pg/mI/day) for 3 days. At the end of the preincubation period, the media were changed to fresh media with or without cGH (100 ng/mI). After an additional 24 h, the supematant was collected, subjected to 10% SDS-PAGE and transferred to nitrocellulose. OPN was detected using a chicken-specific GPN antiserum.

synthesis and differentiation. When cultured in the presence of serum, the cells proliferate (Pines and Hurwitz, 19881, express collagen type II gene (Granot et al., 1993; Fig. lA), low levels of OPN gene (Fig. 1A) and show no AP activity (Halevy et al., 1994; Fig. lB), suggesting that the cells are in their proliferative phase. Addition of ascorbic acid facilitates differentiation, leading to a decrease in cell proliferation (Rosselot et al., 19921, induction of type X collagen (Leboy et al., 1989; Gerstenfeld and Landis, 19911, a decrease

in collagen type II gene expression (Fig. lA), an increase in OPN gene expression (Fig. 1A) and an increase in AP activity (Fig. 1B). In mammals, GH is a major affector of bone growth (for review, see Isaksson et al., 1987; Ohlsson et al., 1993). The hormone interacts directly with its own receptor on the cell surface (Bentham et al., 1993) and augments cell proliferation @ksson et al., 1982; Schlechter et al., 1986; Isaksson et al., 1987). In contrast to mammalian chondrocytes, no increase in

901

48

72

%

Time (b) Fig. 5. Effect of IGF-I and cGH on chondrocyte proliferation. For a long-term serum-free culture, the medium (DMEMI was replaced by Bio-MPM-1 - developed speci&aIIy for anchorage-dependent cells. The cehs were incubated without any addition ( n ) or in the presence of 100 ng/ml IGF-I (0) or cGH (0). At the end of the incubation periods, the media were discarded and the cells were counted using a cell counter. The results are the mean f SE of six separate experiments. The number of cells treated with cGH differ significandy (P < 0.05) from the number of the control cells according to Duncan’s Multiple Range Test.

E. Monsonego et al. /Molecular

and Cellular Endocrinology 114 (1995) 35-42

1000 Conlrol

EGF

haI

LGH

hGH+EGF

cGH+EGF

Fig. 6. Chondrocyte proliferation in the presence of GH and EGF. Cells were incubated either with 20 ng/ml EGF o 100ng/ml GH (human ~^ or chicken), alone or in combination. After 20 h of incubation, [‘Hlthymidine (1 &i/ml) was added for an additional 4 h, after which the DNA-bound [3H]thymidine was determined. The results are the mean f SE of six separate experiments. L3H]Thymidine incorporation to DNA of cells treated with hGH + EGF or cGH + EGF differ significantly (P < 0.05) from all the other treatments according to Duncan’s Multiple Range Test.

cell proliferation was observed after treatment of avian growth plate chondrocytes with cGH (Fig. 51, in agreement with other studies (Rosselot et al., 1992; 1994). Recently we demonstrated that avian growthplate chondrocytes in situ and in culture express the gene coding for the GH-R (Monsonego et al., 1993). In addition, chondrocytes in culture were capable of binding human and chicken GH. Thus, the lack of mitogenic activity of GH on avian chondrocytes was not due to either the absence of GH receptors on the cell membrane or a lack in its activity. Although no increase in cell number was observed after GH treatment, the presence of the hormone was crucial for cell survival in long-term serum-free cultures (Fig. 5). IGF-I, on the other hand, produced a time-dependent increase in cell number as reported for mammalian and avian chondrocytes (Isgaard et al., 1986; Ohlsson et al., 1992b; Rosselot et al., 1994). Although it is generally accepted that the effects of GH on chondrocyte proliferation are mediated via local production of IGF-I in an auto/paracrine manner (Trippel et al., 1987; Isgaard et al., 1988), there is some evidence suggesting that this may not be an exclusive pathway (Loveridge and Farquharson, 1993; Hunziker et al., 1994). The disparity in the responses of avian growthplate chondrocytes to GH and IGF-I in AP activity (Fig. 3) and cell proliferation (Fig. 5) suggests that at least part of the GH action on avian growth-plate chondrocytes is independent of IGF-I production. Although GH by itself did not augment cell proliferation, changes in gene expression, synthesis and activity of various phenotypic markers associated with chondrocyte differentiation were observed. Incubation of chondrocytes with cGH caused an increase in collagen type II gene expression which was time-dependent (Fig. 2A), concomitant with a decrease in the

appearance of OPN in the medium (Fig. 4). In addition, in ascorbic acid-treated cells, GH caused a decrease in AP activity (Fig. 3A). These results suggest that avian growth plate chondrocyte differentiation from proliferative to hypertrophic cells is inhibited by cGH. In mammals, GH promotes prechondrocytes differentiation (Lindahl et al., 1987). It is interesting to note that elevated levels of plasma GH were demonstrated in chickens afflicted with tibia1 dyschondroplasia (TD) (Vasilatos-Younken and Leach, 19861, an abnormality of the growth-plate cartilage which was observed in rapidly growing chickens (Leach and Lilburn, 1992) and turkeys (Wyers et al., 1991). The various morphological and biochemical manifestations of TD lesion, such as changes in carbonic anhydrase and AP activity, type II and X collagen production and OPN gene expression (Gay et al., 1985; Bashey et al., 1989; Chen et al., 1993; Knopov et al., 1995), suggest that TD-chondrocytes fail to undergo complete differentiation which normally leads to cartilage vascularization and mineralization. It is tempting to speculate that elevated levels of GH may interfere with normal growth-plate chondrocyte differentiation leading to TD. The stimulatory effects of GH and IGF-I on mammalian epiphyseal growth plate chondrocytes are related to the differentiated state of the cells. This is supported by the finding that GH but not IGF-I, stimulated the formation of large colonies of cells isolated from the proximal part of the growth-plate (Lindahl et al., 1987). In addition, it has been demonstrated that GH was able to induce cell division in the germinal cell layer in vivo, although no effect of IGF-I was observed (Ohlsson et al., 1992a). These findings served as a basis for the dual-effector theory which implies that GH induces differentiation of vari-

E. Monsonego et al. /Molecular

and Cellular Endocrinology 114 (1995) 35-42

ous mesenchymal progenitor cells, after which IGF-I regulates their clonal expansion (Green et al., 1985; Ohlsson et al., 1993). Other studies challenge this hypothesis by demonstrating that IGF-I and GH were capable of stimulating growth-plate chondrocytes at all stages of differentiation (Hunziker et al., 1994). In our previous study using post-hatched chicken growth-plates, GH-R gene expression was detected both in the proliferative and hypertrophic chondrocytes (Monsonego et al., 1993). Although GH-R gene expression was indistinguishable between chondrocytes at the two stages of differentiation, a disparity in receptor activities was observed. No effect of cGH on type II collagen gene expression (Fig. 2B) or OPN appearance in the medium (Fig. 4) was observed in the ascorbic acid-treated cells. GH was able to reduce AP activity only when added together with ascorbic acid (Fig. 3A) but not after the cells had been previously treated for 4 days with ascorbic acid, when AP activity was at its maximal level. These results are in agreement with our previous findings demonstrating down-regulation of the GH-R gene by GH, only by chondrocytes in their proliferative state (Monsonego et al., 1993). Taken together, we suggest that, in culture, GH can affect only undifferentiated avian growth-plate chondrocytes. Once differentiation occurs, the cells do not respond to the hormone any longer. Chondrocyte proliferation, differentiation and extracellular matrix protein production, are complex processes controlled through the action of several hormones and growth factors. Interactions between the regulatory components must exist to allow precise control of these pathways when cells are exposed to more than a single signal (Rosselot et al., 1994). In avian chondrocytes, growth factor receptors such as FGF-R and EGF-R have been identified (Halevy et al., 1991, 1994). Incubation of chondrocytes with human or chicken GH in combination with EGF produced an increase in chondrocyte proliferation (Fig. 6). The homologous chicken hormone appeared more potent than the heterologous one (Fig. 6), although the chicken hormone binds with an apparently lower affinity to the chicken receptor than does the human hormone (Monsonego et al., 1993). In conclusion, the results suggest that avian chondrocyte differentiation is inhibited by cGH. As a result, the cells remain longer in their proliferative phase and become more susceptible to growth factors such as EGF. Acknowledgements

This study was supported by a grant from BARD, the United States-Israel Binational Agricultural Research and Development Fund (US-2117-92). Contribution from the Agricultural Research Organiza-

tion, The Volcani Center, 1611-E, 1995 series.

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