decreased insulin-like growth factor binding protein-4 (igfbp-4) mrna levels in the liver after...

3
Acta Physiol Scand 1994, 150, 235-237 Decreased i nsul i n-l i ke growth factor binding protein-4 (IGFBP-4) mRNA levels in the liver after hypophysectomy J. PINEDA', R. COYA', G. JACOBSSON', M. LAKE2, K. HALL3 and B. MEISTER' ' Department of Neuroscience, Karolinska Institute, KABI Pharmacia Stockholm, and Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden Insulin-like growth factor (1GF)-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). Almost all tissues that express IGFs and their receptors secrete one or more of the IGFBPs. So far, the hormonal regulation of IGFBP gene expression has only been studied to a very limited extent. Administration of growth hor- mone to hypophysectomized rats causes a rapid increase in I G F I mRNA in the liver in vivo (Norstedt et al. 1987), indicating that hepatic IGF I mRNA expression is regulated by the pituitary gland. Hepatic IGF I1 mRNA levels, on the other hand, do not change after hypophysectomy, whereas IGFBP-2 mRNA expression is increased after this manipulation (Orlowski et a/. 1990). In the present study we have used in situ hybridization to evaluate the effect of hypophysectomy on the expression of IGFBP-4 mRNA in the rat liver. Livers were dissected from control (n = 5) and hypophysectomized (14 days prior to being killed ; n = 5) Sprague-Dawley rats (body wt 150-200 g; Mellegaard Breeding Centre, Copenhagen, Denmark). The livers were then frozen and sectioned at 14 ym in a cryostat. Three complementary 48-mer oligo- Received 19 August 1993, accepted 1 November 1993. Key words: anterior pituitary; growth factor; IGF; in situ hybridization ; regulation Correspondence: Bjorn Meister (M.D., Ph.D.) Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden. nucleotide probes were synthesized (KABI- Pharmacia, Stockholm, Sweden) to nucleotides 484-531, 994-1041 and 1210-1257 of rat IGFBP-4 mRNA (Shimasaki et al. 1990) and purified by HPLC. The probes were labelled with 36S-dATP (NEN, Boston, MA, USA) at the 3'-end using terminal deoxynucleotidyl transferase (IBI, New Haven, CT, USA) and purified using Nensorb 20 columns (NEN). In situ hybridization was performed as described (DagerIind et a/. 1992). Slides were incubated for 16 h at 42 pC with 10" counts min-' of the labelled probes in a solution containing 50% formamide, (1 x SSC = 0.15 M NaCI, 0.015 M sodium-citrate), 1 x Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll and 0.02% polyvinylpyrolidone), 1 yo sarkosyl, 0.02 M NaPO, (pH 7.0), 10% dextran sulphate (pH 7.0), 500mgml-' salmon sperm DNA and 200mM dithiothreitol. After hybridization (16 h), the sections were rinsed in 1 x SSC a 55 "C for 15 min with four changes of SSC and for i 0 min at room temperature, transferred through dis illed water, 60 and 95% ethanol and apposed to L yperfilm-/hax X-ray film (Amersham Ltd., Amersham, UK) at -20 "C. After 2 days of exposure, the film was developed with Kodak LX 24 for 2 min and fixed for 15 min with Kodak AL 4. Measurements were performed on a Macintosh IIx equipped with a frame grabber card, a precision illuminator and a camera. Each image was an average of eight video frames digitized to a 512 x 512 matrix with 256 grey levels for each picture element. The grey levels of eight 14C-plasticstandards (Amersham) exposed to the autoradiography film together with the tissue sections were determined and used in a third degree polynomial approximation to construct a grey level to activity transfer function. The borders of the measuring fields were interactively defined, and the average grey level was calculated using the Macintosh computer and Wingz software together with Cricket Graph. The average grey level of control tissue 23 5 4 x SSC

Upload: j-pineda

Post on 29-Sep-2016

212 views

Category:

Documents


0 download

TRANSCRIPT

Acta Physiol Scand 1994, 150, 235-237

Decreased i nsul i n-l i ke growth factor binding protein-4 (IGFBP-4) mRNA levels in the liver after hypophysectomy

J. PINEDA', R. COYA', G. JACOBSSON', M. LAKE2, K. H A L L 3 and B. MEISTER' ' Department of Neuroscience, Karolinska Institute, KABI Pharmacia Stockholm, and Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden

Insulin-like growth factor (1GF)-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). Almost all tissues that express IGFs and their receptors secrete one or more of the IGFBPs. So far, the hormonal regulation of IGFBP gene expression has only been studied to a very limited extent. Administration of growth hor- mone to hypophysectomized rats causes a rapid increase in IGF I mRNA in the liver in vivo (Norstedt et al. 1987), indicating that hepatic IGF I mRNA expression is regulated by the pituitary gland. Hepatic IGF I1 mRNA levels, on the other hand, do not change after hypophysectomy, whereas IGFBP-2 mRNA expression is increased after this manipulation (Orlowski et a/. 1990). In the present study we have used in situ hybridization to evaluate the effect of hypophysectomy on the expression of IGFBP-4 mRNA in the rat liver.

Livers were dissected from control (n = 5) and hypophysectomized (14 days prior to being killed ; n = 5) Sprague-Dawley rats (body wt 150-200 g; Mellegaard Breeding Centre, Copenhagen, Denmark). The livers were then frozen and sectioned at 14 ym in a cryostat. Three complementary 48-mer oligo-

Received 19 August 1993, accepted 1 November 1993.

Key words: anterior pituitary; growth factor; IGF; in situ hybridization ; regulation

Correspondence: Bjorn Meister (M.D., Ph.D.) Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.

nucleotide probes were synthesized (KABI- Pharmacia, Stockholm, Sweden) to nucleotides 484-531, 994-1041 and 1210-1257 of rat IGFBP-4 mRNA (Shimasaki et al. 1990) and purified by HPLC. The probes were labelled with 36S-dATP (NEN, Boston, MA, USA) at the 3'-end using terminal deoxynucleotidyl transferase (IBI, New Haven, CT, USA) and purified using Nensorb 20 columns (NEN). In situ hybridization was performed as described (DagerIind et a/. 1992). Slides were incubated for 16 h at 42 pC with 10" counts min-' of the labelled probes in a solution containing 50% formamide, (1 x SSC = 0.15 M NaCI, 0.015 M sodium-citrate), 1 x Denhardt's solution (0.02% bovine serum albumin, 0.02% Ficoll and 0.02% polyvinylpyrolidone), 1 yo sarkosyl, 0.02 M NaPO, (pH 7.0), 10% dextran sulphate (pH 7.0), 500mgml-' salmon sperm DNA and 200mM dithiothreitol. After hybridization (16 h), the sections were rinsed in 1 x SSC a 55 "C for 15 min with four changes of SSC and for i 0 min at room temperature, transferred through dis illed water, 60 and 95% ethanol and apposed to L yperfilm-/hax X-ray film (Amersham Ltd., Amersham, UK) at -20 "C. After 2 days of exposure, the film was developed with Kodak LX 24 for 2 min and fixed for 15 min with Kodak AL 4.

Measurements were performed on a Macintosh IIx equipped with a frame grabber card, a precision illuminator and a camera. Each image was an average of eight video frames digitized to a 512 x 512 matrix with 256 grey levels for each picture element. The grey levels of eight 14C-plastic standards (Amersham) exposed to the autoradiography film together with the tissue sections were determined and used in a third degree polynomial approximation to construct a grey level to activity transfer function. The borders of the measuring fields were interactively defined, and the average grey level was calculated using the Macintosh computer and Wingz software together with Cricket Graph. The average grey level of control tissue

23 5

4 x SSC

236 3. Pinedrc et al.

Fig. !(A-D). Film autoradiograms of sections of the rat liver from control (c) and hypophysectomized (hpx) rats after in siru hybridization with an oligo- nucleotide probe to IGFBP-4 mRNA. In control rats, very strong labelling is seen throughout the liver tissues (A). Note a reduction in labelling intensity in the liver after hypophysectomy (A). After hybrid- ization with radiolabelled probe (B) in the presence of 5 (C) and 100 (D) times of excess of cold probe no hybridization signal is observed.

Fig. 2. Effect of hypophysectomy (hpx) on the expression of IGFBP-4 mRNA in the rat liver as compared to controls (c). Levels of mRNA were measured by quantitative autoradiography. There is an almost 14% decrease in IGFBP-4 mRNA levels after hypophysectomy. Statistical analysis was carried out using Mann-Whitney U-test. I*** < 0.005 (n =

5).

sections was set to 100(yo, and changes in grey levels in liver tissue sections from hypophysectomized rats were expressed as a percentage of controls. Statistical analysis was performed using the Mann-Whitney U- test.

In livers from control animals, very strong hybrid- ization with the IGFBP-4 probes was demonstrated in film autoradiograms after 2 days of exposure (Fig. 1 A & B). In emulsion-dipped slides counter-stained with haematoxylin-eosin it could be seen that the labelling was present in hepatocytes (not illustrated). After hypophysectomy there was a decrease in hybridization signal (Fig. 1 A). Quantitative analysis showed that IGFBP-4 mRNA levels in the liver decreased after hypophysectomy with about 14% (P < 0.005) (Fig. 2). No hybridization signal could be observed in the liver after addition of excess (5 or 100 times) of cold probe (Fig. IB, C & D).

The results shows high expression of IGFBP-4 mRNA in the rat liver and its down-regulation after removal of the pituitary gland. A decrease in IGFBP- 4 mRNA after hypophysectomy has recently also been demonstrated in purified Leydig cells (Lin ct ul . 1993).

Future studies may reveal which pituitary hormone that is of importance for regulating the expression of IGFBP-4 mRNA in the liver.

This research was supported by grants from the Swedish Medical Research Council (04X-10358), Stiftelsen Sigurd and Elsa Goljes Minne, Tore Nilsons Stiftelse, Ahlen-stiftelsen, Magnus Bergvalls Stiftelse, and funds from the Karolinska Institute. J . Pineda is

Decreased IGFBP-4 mRNA levels 237

the recipient of a KABI-Pharmacia Research Award. The authors wish to express their gratitude to Ake Dagerlind for help with image analysis.

R E F E R E N C E S

DAGERLIND, A., FRIBERG, K., BEAN, A.J. & HOKFELT, T. 1992. Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry. Histochemistry 98, 39-49.

LIN, T., WANG, D., NAGPAL, M.L., SHIMASAKI, S. & LING, N. 1993. Expression and regulation of insulin-like growth factor binding protein-1, -2, -3, and -4 messenger ribonucleic acids in purified rat leydig cells and their biological effects. Endo- crinology 132, 1898-1904.

NORSTEDT, G., ANDERSON, G., EDWALL, D., ERIKS- SON, L., HANSSON, H.-A., JENNISCHE, E., LEVINO- VITZ, A. & MOLLER, C. 1987. Regulatory aspects of insulin-like growth factor expression in rodents. In :

0. Isaksson, C. Binder, K. Hall & B. Hokfelt (eds), Growth Hormone - Burir and Clinical Aspects, pp. 387-396. Medica International Congress Series 748, Elsevier, Amsterdam.

ORLOWSKI, C.G., BROWN, A.L., 001, G.T., YANG, Y. W.H., TSENG, L.Y .H. & RECHLER, M.M. 1990. Tissue, developmental, and metabolic regulation of messenger ribonucleic acid encoding a rat insulin- like growth factor-binding protein. Endocrinology 126, 644-652.

SARA, V.R. & HALL, K. 1990. Insulin-like growth factors and their binding proteins. Ph,ysrol Ren 70,

SHIMASAKI, S. & LING, N. 1991. Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6). Prog Growth Furtor Res 3, 243-266.

SHIMASAKI, S., USCHIYAMA, F., SHIMONAKA, M. & LING, N. 1990. Molecular cloning of the cDNAs encoding a novel insulin-like growth factor-binding protein from rat and human. Mol Endocrinol 4,

591-614.

145 1-1458.