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.

Abstract

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.

Direct Action of Serotonin on Prolactin, Growth Hormone, Corticotropin and Luteinizing Hormone Release in Cocultures of Anterior and Posterior Pituitary Lobes: Autocrine and/or Paracrine Action of Vasoactive Intestinal Peptide

“…This hypothesis is supported by several studies suggesting that αMSH released from IL cells mediates the PRL release induced by suckling or estrogens [42, 43]or that a fragment of POMC is a growth factor specifically stimulating the development of lactotropes [44]. Moreover, anatomical data support the existence of a diffusible mediator: the area adjacent to the IL is shaped by lactotropes, somatotropes and gonadotropes [45], which are the same cells that respond to 5-HT.…”

Paracrine communication in the anterior pituitary as studied in reaggregate cell cultures

“…One of the factors isolated from GT-conditioned medium capable of increasing the number of 3 H-thymidine incorporating lactotrophs was identified as the 11-kDa N-terminal fragment of proopiomelanocortin (POMC), i.e., POMC or N-POMC (Tilemans et al, 1994). POMC is the well-known precursor molecule that, through proteolytic processing, gives rise to several potent hormones like ACTH, lipotropins, melanotropins, and endorphins.…”

Interaction off αT3-1 Cells with Lactotropes and Somatotropes of Normal Pituitary in vitro

“…A different mechanism may therefore be involved in the action of GnRH on lactotropes and soma totropes. We have presented experimental evidence using pituitary reaggregate cell cultures of 14-day-old female rats that the action of GnRH on PRL secretion as well as on the in vitro differentiation and proliferation of lacto tropes and somatotropes is mediated by paracrine factors released from gonadotropes [8,21,22,28]. As far as secre tion is concerned, the effects were shown not to be due to an artefact of culture [9,18].…”

“…Several of the latter substances were partially purified [21,29]. One was purified to homogeneity and proved to be an 11-kD N-terminal fragment of POMC (POMC,_74) [28], One drawback of the above experimental approach is that the gonadotrope-rich populations used remain con taminated with small numbers of other pituitary cell types, including about 5% corticotropes [28]. Thus, no definite conclusion can be reached as to the cell type pro ducing the above-mentioned factors.…”

“…Another drawback is that the quantity of condi tioned medium collected from primary gonadotropes is, for economical reasons, limited. Still other factors affect ing lactotrope development are present in this medium and the amounts available to identify them were insuffi cient [28,29]. To overcome these problems, it would be of great help to have at our disposal a pure cell line of the gonadotrope lineage which also produces these paracrine factors and from which larger amounts of these factors could be collected.…”