Different Biological Effects of Unmodified Prolactin and a Molecular Mimic of Phosphorylated Prolactin Involve Different Signaling Pathways

Abstract

Previous work has shown that naturally phosphorylated prolactin antagonizes the growth-promoting activities of unmodified prolactin (U-PRL) and that this effect is duplicated by a molecular mimic, S179D PRL. At the same time, the S179D PRL is a superagonist with regard to expression of some PRL-regulated genes. We have asked whether the different activities of U-PRL and S179D PRL are the result of differential signaling. HC11 cells (a normal mouse mammary cell line) were grown to confluence, primed with hydrocortisone, and then exposed to the PRLs. A 15 min incubation of PRL-naive cells led to substantial tyrosine phosphorylation of Jak 2 and Stat 5a by U-PRL and an essentially equivalent Jak 2 activation by S179D PRL. The latter, however, was accompanied by reduced tyrosine phosphorylation of Stat 5a. EMSA analysis using a Stat 5 binding site showed both PRLs to cause equivalent binding of nuclear proteins and that most of what bound was complexed through Stat 5a. Phosphoamino acid analysis of Stat 5 showed S179D PRL to double the amount of serine phosphorylation versus that seen with U-PRL. Analysis of the MAP kinase pathway showed U-PRL capable of activation of ERKs 1 and 2 but that signaling via ERKs 1 and 2 was greater with S179D PRL. A 7-day incubation in either PRL increased beta-casein mRNA levels, but S179D PRL caused a 2-fold increase over that seen with U-PRL. The increase, over that seen with U-PRL, was blocked by the MAP kinase inhibitor, PD98059. After 7 days of treatment with S179D PRL, expression of the short PRL receptor was doubled, and signaling showed a greater dependence on the MAP kinase pathway (2.9-fold increase in ERK 1 and 2 activation). We conclude that although both PRLs use both pathways to some extent, U-PRL signals primarily through Jak 2-Stat 5 whereas S179D PRL signals primarily through the MAP kinase pathway especially after prolonged exposure. This is the first demonstration of differential involvement of signaling pathways by different forms of PRL.

S179D prolactin: Antagonistic agony!

“…In addition, partially phosphorylated PRL (i.e. pituitary extract) showed signaling in between unmodified PRL and S179D PRL, as would be predicted (Coss et al, 1999;Wu et al, 2003). These differences in signaling occurred in cells that up to the initiation of signaling had been treated identically and therefore had the same complement of receptors.…”

“…Thus, one could envisage phosphorylated/S179D PRL simply blocking the effect of unmodified PRL by occupying a receptor, but not inducing a signal. However, as mentioned above, small proportions of phosphorylated PRL/S179D PRL as low as 6-10% have significant effects on cell proliferation Walker,1993,Schroeder et al,2003), Stat 5 activation (Coss et al,1999;Wu et al, 2003;Schroeder et al,2003), and signaling leading to activation of cyclin D1 (Schroeder et al, 2003;Wu et al, 2006). This, as several leading investigators said at the time of our original presentation of this finding, is impossible!…”

A mimic of phosphorylated prolactin induces apoptosis by activating AP-1 and upregulating p21/waf1 in human prostate cancer PC3 cells

“…Nevertheless, a short mouse PRLR was able to activate mitogen-activated protein kinases (MAPKs), which led to the proliferation of NIH/3T3 cells (3). Binart et al revealed that a short form of mouse PRLR rescues mammopoiesis in heterozygous PRLR mice (4). Notably, we demonstrated that S179D PRL induced β-casein gene expression by regulating the ratio of short to long PRLR in mouse mammary HC11 cells (5).…”

S179D prolactin diminishes the effects of UV light on epidermal gamma delta T cells

“…Oetting et al,1986;Wu et al, 2003) and so this is one potential source of this form of the hormone, with delivery to the skin via the circulation. However, PRL has also been demonstrated to be produced in the skin where it can act as an autocrine/paracrine factor (Foitzik et al, 2006).…”

“…However, studies in which the two major forms of PRL have been individually analyzed have shown them to have very different biological functions. Some of these studies used the naturally phosphorylated material (Krown et al, 1992, Wang and, while others utilized a stable molecular mimic of phosphorylated PRL, S179D PRL (Chen et al, 1998;Xu et al, 2001;Yang et al, 2001;Kuo et al,2002;Yang et al, 2002;Schroeder et al, 2003;Wu et al, 2003;Xu et al, 2003;Naylor et al, 2005;Guzman et al, 2005;Ueda et al, 2006a,b;Wu et al 2006). In the current study, we assessed the effects of either U-PRL or S179D PRL on LC and γδT in mature skin, in the absence or presence of a suberythemal dose of UVB.…”