Analysis of the Proteolytic Cleavage of Prolactin by the Mammary Gland and Liver of the Rat: Characterization of the Cleaved and 16K Forms*

Estrogens are recognized regulators of the expression of neurohypophyseal hormones and of anterior pituitary prolactin (PRL). Here we have investigated whether the levels of PRL mRNA and of 23 and 14 kDa PRL variants present in the hypothalamo-neurohypophyseal system change during the estrous cycle or in response to estrogen treatment. The reverse transcription polymerase chain reaction (RT-PCR) was performed to examine PRL mRNA expression in isolated paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei. In both nuclei PRL mRNA levels appeared higher in cycling females than in male rats, with the highest level occurring at estrus. This increase may involve estrogen action, since estrogen administration to ovariectomized rats was associated with apparently higher PRL mRNA levels in both the PVN and SON. Expression of the PRL gene at these sites may occur via both transcriptional factor Pit-1-dependent and -independent mechanisms. RT-PCR detected the mRNA for Pit-1 in the PVN but only at estrus. The concentration of the 23 kDa immunoreactive PRL determined in the neurohypophysis was significantly higher during estrus and after estrogen treatment. However, no difference was detected in the levels of the neurohypophyseal 14 kDa PRL-like fragment along the estrous cycle nor after estrogen administration. This lack of parallelism between neurohypophyseal PRLs could relate to an estrogen-induced inhibition of the proteolysis of 23 kDa PRL at this site, since estrogen treatment reduced the activity of neurohypophyseal proteolytic enzymes able to cleave PRL. Altogether our results are consistent with estrogens having a stimulatory effect on PRL gene expression in the hypothalamo-neurohypophyseal system and a concomitant inhibitory action on PRL proteolysis at this site.

Section: Prl-cleaving Activitymentioning

confidence: 77%

“…The reported procedure with the following modifications was used (Clapp et al 1987). Briefly, neurointermediate lobes were homogenized in cold 0·1 M Tris-HCl/0·25 M sucrose (pH 7·4; 1 µg/50 µl) and microfuged (14 000 g) for 10 min at 4 C. The resulting pellet was resuspended in 0·1 M Tris-HCl (pH 7·4).…”

Section: Analysis Of Prl-cleaving Activitymentioning

confidence: 99%

Changes in the expression of neurohypophyseal prolactins during the estrous cycle and after estrogen treatment

Torner¹,

Nava²,

Dueñas³

et al. 1999

“…16K PRL was generated after the enzymatic proteolysis of PRL with a particulate fraction from rat mammary glands, gel filtration, and carbamidomethylation, as described (Clapp 1987).…”

Section: Methodsmentioning

confidence: 99%

16K prolactin induces NF-kappaB activation in pulmonary fibroblasts

Macotela¹,

Mendoza²,

Corbacho³

et al. 2002

The amino-terminal 16 kDa fragment of prolactin (16K PRL) promotes the expression of the inducible isoform of nitric oxide synthase (iNOS) accompanied by the production of nitric oxide (NO) by rat pulmonary fibroblasts. The present study was designed to elucidate whether the mechanism by which 16K PRL promotes iNOS expression involves the activation of nuclear factor-kappa B (NF-B), a key transcription factor for iNOS induction. 16K PRL stimulated DNA-binding activity of NF-B in pulmonary fibroblasts as demonstrated by gel shift assays. Likewise, fluorescence immunocytochemistry showed that 16K PRL promotes nuclear translocation of the p65 subunit of NF-B. Finally, treatment with 16K PRL induced the degradation of the NF-B inhibitor B-beta (I B-), and such degradation was prevented by blocking I B-phosphorylation. Altogether, these results show that 16K PRL activates NF-B nuclear translocation via the phosphorylation and degradation of I B-. These findings are consistent with NF-B being part of the signal transduction pathway activated by 16K PRL to induce iNOS expression.

“…In addition, circulating PRL may be proteolytically processed to 16K PRL in target tissues (Tables 1 and 2). In this regard, PRL cleaving activity has been demonstrated in homogenates of mammary gland (Wong et al 1986, Clapp 1987), brain (DeVito et al 1992, Clapp et al 1994, posterior pituitary (Clapp et al 1994), prostate, liver, kidney and spleen (Compton & Witorsch 1984, Clapp 1987, Baldocchi et al 1992. Finally, several extra-pituitary cell types express the PRL gene (Ben-Jonathan et al 1996) and can cleave locally produced PRL.…”

Section: Endogenous 16k Prolactinmentioning

confidence: 99%

Roles of prolactin and related members of the prolactin/growth hormone/placental lactogen family in angiogenesis

Corbacho¹,

Escalera²,

Clapp³

2002

234

149

Prolactin, growth hormone and placental lactogen are members of a family of polypeptide hormones which share structural similarities and biological activities. Numerous functions have been attributed to these hormones, among which stand out their recently discovered effects on angiogenesis, the process by which new blood vessels are formed from the pre-existing microvasculature. Prolactin, growth hormone and placental lactogen, along with two non-classical members of the family, proliferin and proliferin-related protein, can act both as circulating hormones and as paracrine/autocrine factors to either stimulate or inhibit various stages of the formation and remodeling of new blood vessels, including endothelial cell proliferation, migration, protease production and apoptosis. Such opposing actions can reside in similar but independent molecules, as is the case of proliferin and proliferin-related protein, which stimulate and inhibit angiogenesis respectively. The potential to exert opposing effects on angiogenesis can also reside within the same molecule as the parent protein can promote angiogenesis (i.e. prolactin, growth hormone and placental lactogen), but after proteolytic processing the resulting peptide fragment acquires anti-angiogenic properties (i.e. 16 kDa prolactin, 16 kDa growth hormone and 16 kDa placental lactogen). The unique properties of the peptide fragments versus the full-length molecules, the regulation of the protease responsible for specific protein cleavage, the selective expression of specific receptors and their associated signal transduction pathways are issues that are being investigated to further establish the precise contribution of these hormones to angiogenesis under both physiological and pathological situations. In this review article, we summarize the known and speculative issues underlying the effects of the prolactin, growth hormone and placental lactogen family of proteins on angiogenesis, and address important remaining enigmas in this field of research.