Acta Physiol Scand 1994, 150, 345-346

Chondrocytes of the adult rat express mRNA for insulin-like growth factor binding protein-4 ( IG F B P-4)

J. PINEDA,' R. COYA,' G. J A C O B S S O N , ' M. L A K E , ' K. H A L L 3 and B. MEISTER' ' Department of Neuroscience, Karolinska Institute, KABI Pharmacia and Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden

Growth hormone (GH) is the major hormone that regulates longitudinal bone growth in a dose-de- pendent manner. However, studies have also shown that insulin-like growth factor (1GF)-I plays an important role in normal bone growth (Isaksson et al. 1987). IGF-I and -11 are mitogenic polypeptides, which in plasma and other biological fluids are associated with specific binding proteins (IGFBPs) (Sara & Hall 1990). In addition to their role as carrier proteins for IGFs, the IGFBPs can modulate the biological activity of IGFs. At least six different IGFBP cDNAs have been cloned and their complete primary structures have been deduced from the cDNA clones in both rat and human species (Shimasaki & Ling 1991). Recently, IGF-1 binding to rat tibia1 epiphyseal chondrocytes was demonstrated (Bentham et al. 1993), suggested the production of IGFBP in cartilage. In the present study we have used in situ hybridization to study the localization of IGFBP-4 mRNA in cartilage of the rat rib.

Ribs from SpragueDawley rats (body wt 150-200 g; ALAB, Sollentuna, Sweden), frozen and sectioned at 14 pm in a cryostat. Three complementary 48-mer oligonucleotide probes were synthesized (KABI-Pharmacia, Stockholm, Sweden) to nucleo- tides484-531,994-1041 and 1210-1257ofrat IGFBP- 4 mRNA (Shimasaki et al. 1990) and purified by HPLC. The probes were labelled with 9 - d A T P (NEN, Boston, MA, USA) at the 3'-end using terminal deoxynucleotidyl transferase (IBI, New Haven, NJ, USA) and purified using Nensorb 20 columns (NEN). I n situ hybridization was performed as described (Dagerlind et al. 1992). Slides were

Received 13 October 1993, accepted 18 November 1993.

Key words : cartilage, chondrocyte, growth factor, IGF, IGFBP, in situ hybridization, rib.

Correspondence : Bjorn Meister, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden.

incubated for 16 h at 42 "C with 10'cpm of the labelled probes in a solution containing 50% form- amide, 4 x SSC (1 x SSC = 0.15 M NaCI, 0.015 M sodium citrate), 1 x Denhardt's solution (0.02%

Fig. 1 (a<). I n situ hybridization of adjacent sections of the rat rib using an oligonucleotide probe to IGFBP-4 mRNA. In the film autoradiogram (a), hybridization signal is present in the peripheral part of the hyaline cartilage, whereas the central part is devoid of labelling. In an emulsion-dipped and haematoxylin-counterstained section (b, c), silver grains can be detected overlying chondrocytes close to the perichondrium (pc) as well as in chondrocytes organized in isogenic groups (g) (see arrows). Bar in a and b = 1 mm. Bar in c = 100pm.

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bovine serum albumin, 0.02% Ficoll and 0.0270 polyvinylpyrrolidone), 1 yo sarkosyl, 0.02 M NaPO, (pH 7.0), 10% dextran sulphate (pH 7.0), 500 yg ml-' salmon testis DNA and 200 mM dithio- threitol. After hybridization (16 h), the sections were rinsed in 1 x SSC at 55 "C for 15 min with four changes of SSC and for 60min at room temperature, transferred through distilled water, 60% and 95% ethanol and apposed to Hyperfilm-/hax autoradiography film (Amersham Ltd., Amersham, UK) at -20 "C. After 8 days of exposure, the film was developed with Kodak LX 24 for 2 min and fixed for 15 min with Kodak AL 4. Some sections were dipped in Kodak NTB2 emulsion, exposed for 4 weeks at - 20 "C, developed in Kodak D19 and fixed in Kodak 3000, and thereafter counterstained with haematoxylin-eosin and cover- slipped with Entellan@. All sections were examined under bright- or dark-field illumination using a Nikon Microphot-FX microscope. Photomicrographs were taken with Kodak T-Max 100 film.

In film autoradiograms, hybridization signal could be seen in the periphery of hyaline costal cartilage of the rat, whereas the central part was devoid of labelling (Fig. 1 a). After emulsion-dipping and subsequent counter-staining with haematoxylin-eosin (Fig. 1 b, c), silver grains were detected over chondro- cytes located close to the perichondrium and organized in isogenic groups (Fig. 1 c). Identical labelling patterns were observed with all three probes (not shown). No hybridization signal could be detected after addition of excess (100 times) of cold probe to the radiolabelled probe (not shown).

These results show that IGFBP-4 mRNA is expressed in chondrocytes of the adult rat. IGFBP-4, -5 and -6 mRNA have been detected in ribs of the fetal mouse (Schuller et al. 1993), however, cellular localization has not been determined. The presence of IGFBP-4 mRNA in chondrocytes suggests that IGFBP-4 together with G H and IGF may play a role in the regulation of somatic growth. Further studies may reveal the role of IGFBP-4 in regulation of

longitudinal bone growth and the regulation of IGFBP-4 mRNA expression in bone.

This research was supported by grants from the Swedish Medical Research Council (04X-10358), Tore Nilsons Stiftelse, Ahlin-stiftelsen, Magnus Bergvalls Stiftelse, and funds from the Karolinska Institute. J. Pineda is the recipient of a KABI-Pharmacia Research Award.

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