In vitro evidence that an 11-kilodalton N-terminal fragment of proopiomelanocortin is a growth factor specifically stimulating the development of lactotrophs in rat pituitary during postnatal life.
We have previously shown that in reaggregate cell cultures of 14-day-old female rat pituitary, LHRH is capable of stimulating lactotrophs to enter the S-phase of the cell cycle, and that this effect is mediated by paracrine growth factors secreted from gonadotrophs. In the present report we describe the isolation, purification, and chemical characterization of one of these growth factors. Gonadotroph-rich aggregates from 14-day-old female rats were cultured for 6-7 weeks in serum-free and serum albumin-free defined medium in the presence of 0.01 nM LHRH. A total volume of 3.2 liters containing material secreted from 2.5 x 10(8) cells was batchwise concentrated on Sep-Pak C18/125 A cartridges (10 g) and retained molecules ultrafiltrated on Centricon 3 membrane filters [mol wt (M(r)) cut-off, 3 kilodaltons (kDa)]. Material capable of increasing the number of [3H]thymidine ([3H]T)-incorporating lactotrophs in pituitary cell aggregates of 2-week-old rats was isolated by chromatography on two reverse phase HPLC columns, one HPLC gel filtration column, and a final reverse phase HPLC column. A substance was obtained which, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, ran as a single band with an apparent M(r) of 16 kDa. On gel filtration, the apparent M(r) was 11 kDa. N-Terminal amino acid sequence analysis revealed that the substance was a peptide; a sequence of 10 residues was obtained, which was identical to that in the N-terminal part of rat POMC. By electrospray ionization mass spectrometry, six different compounds with slightly different M(r), ranging from 10,796-11,108 daltons, were detected. The latter data suggest that the peptide extends C-terminally at least to residue 74, which in the POMC sequence is flanked by an Arg-Arg dibasic residue, a posttranslational cleavage site. The substance increased the number of [3H]T-incorporating lactotrophs in pituitary cell aggregates without affecting [3H]T labeling of other pituitary cell types. Authentic human POMC-(1-76), at a concentration of 10 nM provoked a similar stimulation of the [3H]T-labeled lactotroph number without affecting other cell types. It is concluded that POMC-(1-74) is a growth factor that specifically targets lactotrophs during postnatal development of the rat anterior pituitary.
Department of Molecular Cell Biology, University of Leuven School of Medicine, Belgium.
D
D
Tilemans
M
M
Andries
P
P
Proost
B
B
Devreese
J
J
Van Beeumen
C
C
Denef
D000818:Animals; D000831:Animals, Newborn / Q000254:growth & development*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006133:Growth Substances / Q000494:pharmacology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology*; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000737:chemistry* / Q000494:pharmacology*; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D006133:Growth Substances; D010446:Peptide Fragments; D011333:Pro-Opiomelanocortin; D011388:Prolactin