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D Swolin-Eide and C Ohlsson

High levels of glucocorticoids are believed to alter bone remodeling by decreasing bone formation and increasing bone resorption. It has been suggested that different cytokines, like interleukin-6 (IL-6) and interleukin-1 (IL-1), are involved in bone resorption by activating immature osteoclasts, and some studies indicate that IL-6 promotes bone formation by a mitogenic effect on osteoblasts. The aim of the present investigation was to study whether cortisol regulates the expression of IL-6 and IL-1 beta in human osteoblast-like cells. A high dose of cortisol (10(-7)M) decreased, as expected, the C-terminal propeptide of type I collagen released into the culture medium. The IL-6 mRNA levels and IL-6 protein released into the culture medium were also decreased by cortisol in a dose-dependent manner. The maximum effect was seen at 1 microM cortisol (mRNA 23.1 +/- 7.9% of control culture; protein 28.2 +/- 8.3% of control culture). The decrease in IL-6 mRNA levels was apparent 4 h after the addition of cortisol and was still present 20 h later. The decrease in IL-6 protein released into the culture medium was seen 20 h later than the decrease in IL-6 mRNA levels. The production of IL-1 beta protein released into the culture medium was decreased in a dose-dependent manner after the addition of cortisol with a maximum effect at 1 microM. The effect of cortisol on IL-1 beta protein released into the culture medium was seen 16 h after the addition of cortisol. To summarize, cortisol decreases the expression of IL-6 as well as IL-1 beta in human osteoblast-like cells.

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D Swolin-Eide, A Nilsson and C Ohlsson

It is well known that high levels of glucocorticoids cause osteoporosis and that physiologic levels of growth hormone (GH) are required for normal bone remodeling. It has been suggested that glucocorticoids regulate GH-responses via the regulation of GH-receptor expression. The aim of the present study was to investigate whether cortisol plays a role in the regulation of GH-receptor expression in cultured human osteoblasts. The effect of serum starvation and cortisol on GH-receptor expression was tested in human osteoblast (hOB)-like cells. Serum starvation for 24 h resulted in an increase in GH-receptor mRNA levels (90 +/- 1% over control culture). Cortisol increased GH-receptor mRNA levels in a dose-dependent manner with a maximal effect at 10(-6)M. The stimulating effect of cortisol on GH-receptor mRNA levels was time-dependent, reaching a peak 12 h after the addition of cortisol (126 +/- 29% over control culture) and remaining up to 12 h later. The increase in GH-receptor mRNA levels was accompanied by an increase in 125I-GH binding which reached a maximum at 24 h (196 +/- 87% over control culture). In conclusion, glucocorticoids increase GH-receptor expression in hOB-like cells. Further studies are needed to clarify whether glucocorticoid-induced regulation of the GH-receptor is important in human bone physiology.

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D Swolin, C Brantsing, G Matejka and C Ohlsson

Abstract

Excess levels of glucocorticoids are known to cause osteoporosis. It is speculated that the effect of glucocorticoids could be mediated via regulation of IGF-I. The aims of the present study were to detect and quantify the expression of IGF-I and/or IGF-II mRNA transcripts in human osteoblast-like cells and to investigate whether glucocorticoids regulate the expression of IGF-I mRNA transcripts in human osteoblast-like cells.

Cultures of human osteoblast-like cells from trabecular bone were established. The IGF-IA and IGF-IB transcripts were detected in human osteoblast-like cells from seven out of nine patients while the IGF-II transcript was detected in human osteoblast-like cells from eight out of nine patients, as determined by RT-PCR assays. Human osteoblast-like cells, as well as human muscle tissue, expressed approximately 1/10 of the IGF-I mRNA levels found in liver, as determined by RNase protection solution hybridization assay. The IGF-I mRNA levels did not decrease with age in the human osteoblast-like cells and no difference was seen between males and females. However, cortisol (10−6 mol/l) decreased IGF-I mRNA levels.

In summary, the present study has shown that human osteoblast-like cells express IGF-I and IGF-II mRNA transcripts and that cortisol down-regulates the IGF-I mRNA levels, indicating that some of the inhibitory effect of glucocorticoids on bone formation in humans is mediated via a reduced autocrine/paracrine expression of IGF-I.

Journal of Endocrinology (1996) 149, 397–403

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J. Bentham, C. Ohlsson, A. Lindahl, O. Isaksson and A. Nilsson

ABSTRACT

In the present study a double-staining technique was developed to investigate simultaneous GH and insulin-like growth factor-I (IGF-I) binding to chondrocytes in a monolayer cell culture.

Rat tibial epiphyseal chondrocytes were isolated by enzymatic digestion and cultured in monolayer. GH and IGF-I were labelled with biotin. The affinity of the biotin-labelled ligands was compared with unlabelled ligands in a radioreceptor assay.

To study the distribution of GH and IGF-I binding in the monolayer, chondrocytes were incubated with biotinylated ligands with or without an excess of unlabelled ligands, followed by incubation with Vectastain ABC complex, which was then reacted with diaminobenzidine (DAB). Double staining was accomplished by carrying out the first reaction with DAB in the presence of nickel ammonium sulphate to give a black precipitate, followed by incubation with the second ligand, then ABC complex and finally DAB in the absence of nickel ammonium sulphate to give a brown stain. The presence of type-II collagen was demonstrated by immunohistochemistry and used as a marker for differentiated chondrocytes.

Biotin-labelled GH and biotin-labelled IGF-I exhibited dose-dependent displacements of 125I-labelled GH and 125I-labelled IGF-I respectively from the chondrocytes in a radioreceptor assay. The displacement curves were identical to those of unlabelled ligands indicating that the affinity was unaltered. Binding of biotinylated GH to cells was seen throughout the culture in regions where there was little or no type-II collagen staining. IGF-I binding was predominantly localized to cells at high density; areas which also showed a high degree of staining for type-II collagen.

The different locations of binding suggest that epiphyseal chondrocytes in monolayer culture comprise a heterogeneous cell population and that IGF-I and GH have different target cells.

Journal of Endocrinology (1993) 137, 361–367

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N Andersson, MK Lindberg, C Ohlsson, K Andersson and B Ryberg

The recent development of different genetically modified mice with potentially interesting bone phenotypes has increased the demand for effective non-invasive methods to evaluate effects on bone of mice during growth and development, and for drug evaluation. In the present study, the skeleton was analyzed by repeated in vivo scans using dual energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Ovariectomized (ovx) mice treated with parathyroid hormone (PTH) were used as an animal model to evaluate these two techniques at different times after the onset of treatment. Female mice (6 weeks of age) were allocated randomly to four groups: (1) sham-operated+vehicle; (2) ovx+vehicle; (3) sham-operated+PTH(1-84) 150 microg/kg per day; (4) ovx+PTH. Six weeks after ovariectomy the drug treatment began and was continued for 8 weeks. The total body bone mineral content (BMC) and total body areal bone mineral density (BMD) were measured by DXA. Ovariectomy reduced total body BMC and total body areal BMD by 6.2+/-1.7% and 2.6+/-0.9% respectively. No effect of PTH on total body BMC was seen during the treatment period. The trabecular volumetric BMD was measured by pQCT. Ovariectomy reduced the trabecular volumetric BMD by 52+/-6.7%. The pQCT technique detected a clear effect on trabecular volumetric BMD after 2 weeks of PTH treatment (ovx 94+/-29% and sham-operated 46+/-10% more than vehicle-treated). The cortical bone was measured in a mid-diaphyseal pQCT scan of the tibia. Ovariectomy reduced the cortical BMC by 9+/-2%. PTH treatment for 8 weeks increased cortical BMC in ovx mice. In conclusion, the pQCT technique is more sensitive than the DXA technique in the detection of bone loss after ovariectomy and increased bone mass after PTH treatment in mice. Notably, the pQCT, but not the DXA, technique detected a dramatic effect as early as after 2 weeks of PTH treatment. Dynamic pQCT measurements will be useful for monitoring skeletal changes during growth and development, and for drug evaluation in mice.

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JM Kindblom, O Nilsson, T Hurme, C Ohlsson and L Savendahl

Indian Hedgehog (Ihh) has been reported to control the rate of cartilage differentiation during skeletal morphogenesis in rodents through a negative feedback loop involving parathyroid hormone related protein (PTHrP). The role of Ihh and PTHrP in the regulation of human epiphyseal chondrocytes is unknown. The aim of the current study was to examine the expression and localization of Ihh and PTHrP in the human growth plate at various pubertal stages. Growth plate biopsies were obtained from patients subjected to epiphyseal surgery and the expression of Ihh and PTHrP was detected by immunohistochemistry. We show that Ihh and PTHrP are expressed mainly in early hypertrophic chondrocytes in the human growth plate. The levels of expression of Ihh and PTHrP are higher in early stages of puberty than later. Our results suggest that Ihh and PTHrP are present in the human growth plate and that Ihh and PTHrP may be involved in the regulation of pubertal growth in humans.

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C. Ohlsson, A. Nilsson, O. Isaksson, J. Bentham and A. Lindahl

ABSTRACT

The effects of tri-iodothyronine (T3) and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation, alkaline phosphatase (ALP) activity and IGF-I receptor mRNA levels were studied in rat epiphyseal chondrocytes cultured in monolayer.

Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in monolayer culture and precultured for 7–14 days in Ham's F-12 medium supplemented with 10% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture the medium was changed to Ham's F-12 medium containing 1% (v/v) serum from hypophysectomized rats, and the effects of T3 and/or IGF-I on DNA synthesis ([3H]thymidine incorporation), ALP activity (a late marker of differentiated epiphyseal chondrocytes) and IGF-I receptor mRNA levels were studied.

ALP activity was increased by T3 in a dose-dependent manner with a maximal response at 10 μg T3/1 (678 ±86% compared with control culture). The increase in ALP activity was accompanied by a concomitant decrease in [3H]thymidine incorporation (52 ±14% compared with control culture). Human GH (hGH; 50 μg/l) and IGF-I (25 μg/l) had no stimulatory effect on ALP activity. However IGF-I (10 μg/l) exerted an inhibition on the T3 (10 μg/l)-induced increase in ALP activity (64 ± 9% compared with T3-treated culture). T3 (3 μg/l) inhibited the increase in [3H]thymidine incorporation caused by 25 μg IGF-I/l(51 ± 13% compared with IGF-I-treated culture). Furthermore, IGF-I receptor mRNA levels were increased by 10 μg T3/l (137 ±4·2% compared with control culture) while no effect of hGH (50 μg/l) or IGF-I (25 μg/l) was demonstrated.

Both T3 and IGF-I were shown to interact with epiphyseal chondrocytes and both substances seemed to affect cell proliferation and maturation and therefore longitudinal bone growth. Furthermore, the results indicated that IGF-I is important for proliferation of the cells while T3 initiates the terminal differentiation of epiphyseal chondrocytes.

Journal of Endocrinology (1992) 135, 115–123

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T Ahmad, C Ohlsson, M Saaf, CG Ostenson and A Kreicbergs

We characterized appendicular and axial bones in rats with type-2 diabetes in five female Goto-Kakizaki (GK) rats, a strain developed from the Wistar rat showing spontaneous type-2 diabetes, and five age- and sex-matched non-diabetic Wistar rats. The humerus, tibia, metatarsals and vertebral bodies were analysed by peripheral quantitative computerized tomography (pQCT). In diabetic rats, the height of the vertebral bodies and length of the humerus were decreased while the length of the metatarsals was increased. A decreased cross-sectional area was found in the vertebral end-plate region and the tibial metaphysis. Notably, the diaphysis in all long bones showed expansion of periosteal and endosteal circumference. In tibia this resulted in increased cortical thickness, whereas in humerus and metatarsal it was unchanged. Areal moment of inertia was increased in all diaphyses suggesting greater bending strength. The most conspicuous finding in diabetic rats pertained to trabecular osteopenia. Thus, trabecular bone mineral density was significantly reduced in all bones examined, by 33-53%. Our pQCT study of axial and appendicular bones suggests that the typical feature of diabetic osteopathy in the GK rat is loss of trabecular bone and expansion of the diaphysis. The loss of metaphyseal trabecular bone if also present in diabetic patients may prove to underlie the susceptibility to periarticular fracture and Charcot arthropathy. The findings suggest that the risk of fracture in diabetes varies according to the specific sub-regions of a bone. The approach described may prove to be useful in the early detection of osteopathy in diabetic patients who may be amenable to preventive treatment.

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NO Vidal, S Ekberg, S Enerback, A Lindahl and C Ohlsson

The transcription factor C/EBP alpha, a member of the CCAAT/enhancer-binding protein family, is highly expressed in the liver and in adipose tissue. The aim of this study was to determine if C/EBP alpha is expressed in rat growth cartilage. The expression pattern of C/EBP alpha in monolayer-cultured growth plate chondrocytes was similar to that of C/EBP alpha during hepatocyte and preadipocyte differentiation. Immunohistochemistry with a polyclonal antibody for C/EBP alpha revealed that the C/EBP alpha protein is present in the perichondrial ring, in the germinal layer of the growth plate and on the surface of the articular cartilage. The growth hormone (GH) receptor has a similar distribution in the rat tibial growth plate, and hypophysectomised rats were used to investigate a possible connection between C/EBP alpha and GH. C/EBP alpha mRNA levels were decreased in rib cartilage after hypophysectomy. However, GH treatment did not counteract this effect, indicating that other pituitary hormones regulate the C/EBP alpha mRNA levels in growth plate cartilage. We thus demonstrate, for the first time, that C/EBP alpha is expressed in cartilage. The finding that C/EBP alpha, like the GH receptor, is predominantly expressed in stem cell areas of the rat growth plate indicates a possible functional role for C/EBP alpha during early chondrogenic differentiation.

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C. Ohlsson, A. Nilsson, O. G. P. Isaksson and A. Lindahl

ABSTRACT

The influence of various culture conditions was studied on the effect of GH and insulin-like growth factor-I (IGF-I) on DNA and matrix synthesis in epiphyseal rat chondrocytes in monolayer culture.

Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in 48-well culture plates and precultured for 10 days in Ham's F-12 medium supplemented with 1% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture, the medium was changed to Ham's F-12 medium supplemented with 1% serum from hypophysectomized rats, and the effect of GH and IGF-I on DNA synthesis ([3H]thymidine incorporation) and matrix production ([35S]sulphate uptake) was studied during an additional 96-h culture period. Isotopes were present during the last 24 h of culture.

Both hGH and IGF-I stimulated DNA synthesis in a dose-dependent manner. A maximal effect of GH was seen at a concentration of 25 μg/l (60 ± 11% stimulation over control) and for IGF-I at 10 μg/l (162 ± 12%). The stimulatory effects of the same concentrations of human GH (hGH) and IGF-I on [35S]sulphate uptake were 135 ± 25 and 320 ± 42% respectively. In-vitro pulse labelling revealed that GH did not produce a response during the first 3 days of culture (after addition of GH) but was effective during days 4 and 5 of culture. In contrast, IGF-I was effective throughout the culture period. Pretreatment of cells with GH or IGF-I for 2·5 days showed that GH but not IGF-I produced a sustained effect on [3H]thymidine uptake.

In order to study the influence of cell density on the effect of GH and IGF-I on DNA synthesis, the effect of added peptides was evaluated after different preculture periods (5–15 days). A maximal stimulatory effect of hGH was seen at a cell density of 150 000–300 000 cells/cm2. GH had no significant effect at a low (< 100 000 cells/cm2) or a high (>400 000 cells/cm2) cell density. The magnitude of the stimulatory effect of IGF-I was the same at densities between 10 000 and 250 000 cells/cm2, but was reduced at higher cell densities (over 250 000 cells/cm2).

Chondrogenic properties of cells that had been cultured for 15 days were verified in vitro by positive alcian blue staining and identification of type II collagen, and in vivo by development of cartilage nodules in nude mice.

The results from the present study clearly show that GH and IGF-I both stimulate DNA synthesis and matrix production in epiphyseal chondrocytes in monolayer culture. The results also demonstrate that expression of the effect of GH is highly dependent upon the culture conditions.

Journal of Endocrinology (1992) 133, 291–300