Leukemia Inhibitory Factor–dependent Transcriptional Activation in Embryonic Stem Cells

Abstract

STAT transcription factors are induced by a number of growth factors and cytokines. Within minutes of induction, the STAT proteins are phosphorylated on tyrosine and serine residues and translocated to the nucleus, where they bind to their DNA targets. The leukemia inhibitory factor (LIF) mediates pleiotropic and sometimes opposite effects both in vivo and in cultured cells. It is known, for example, to prevent differentiation of embryonic stem (ES) cells in vitro. To get insights into LIF-regulated signaling in ES cells, we have analyzed protein-binding and transcriptional properties of STAT recognition sites in ES cells cultivated in the presence and in the absence of LIF. We have detected a specific LIF-regulated DNA-binding activity implicating the STAT3 protein. We show that STAT3 phosphorylation is essential for this LIF-dependent DNA-binding activity. The possibility that ERK2 or a closely related protein kinase, whose activity is modulated in a LIF-dependent manner, contributes to this phosphorylation is discussed. Finally, we show that the multimerized STAT3-binding DNA element confers LIF responsiveness to a minimal thymidine kinase promoter. This, together with our observation that overexpression of STAT3 dominant-negative mutants abrogates this LIF responsiveness, clearly indicates that STAT3 is involved in LIF-regulated transcriptional events in ES cells. Finally, stable expression of such a dominant negative mutant of STAT3 induces morphological differentiation of ES cells despite continuous LIF supply. Our results suggest that STAT3 is a critical target of the LIF signaling pathway, which maintains pluripotent cell proliferation.

LIF-Induced STAT3 Signaling in Murine versus Human Embryonal Carcinoma (EC) Cells

“…As a consequence, mouse ES cells can now be cultured in the absence of embryonal fibroblast feeder-cell layers when LIF is administered to the medium [5][6][7][8]. However, human ES cells do not respond to LIF in a similar fashion and still require feeder-cell layers for support [27].…”

“…Mouse ES cells can be maintained in an undifferentiated state when cultured on fibroblast feeder cell layers or in the presence of leukemia inhibitory factor (LIF) on gelatin-coated tissue culture dishes [5][6][7][8]. LIF belongs to the interleukin (IL)-6 family of cytokines, which also includes oncostatin M, ciliary neurotropic factor, IL-11, cardiotrophin, and IL-6 [9].…”

“…The pluripotency of mouse stem cells in culture depends on LIF-activated STAT3, since mutation of either the gp130 receptor or STAT3 itself abrogated the self-renewal of ES cells and led to the onset of differentiation [5,7,8]. Consequently, mouse stem cells can be cultured and kept pluripotent on gelatin-coated tissue culture flasks when maintained in medium containing LIF.…”

Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1

“…In M1 cells, at least, IFNg-responses which depend on Jak1 and Jak2 are not a ected by the overexpression of dominant negative STAT3 . Also in other cellular systems STAT3 is essential for the function of IL-6-type cytokines: for the survival of transfected BAF ± B03 pro-B-cells , for the di erentiation of promyelocytic M1 cells to macrophage-like cells (Minami et al, 1996;Nakajima et al, 1996;Yamanaka et al, 1996) and for the proliferation and maintenance of pluripotency of embryonic stem cells (Boeuf et al, 1997;Niwa et al, 1998). However, not all IL-6 e ects depend on STAT3: neurite outgrowth of PC12 pheochromocytoma cells needs signals via Y759 leading to activation of the tyrosine phosphatase SHP-2 and in turn to MAP kinase activation.…”

“…Myeloid M1 cells required functional STAT3 to di erentiate into macrophage-like cells (Minami et al, 1996;Nakajima et al, 1996). The inhibition of differentiation and maintenance of pluripotency of embryonic stem cells cultured in the presence of LIF similarly depended on STAT3 (Boeuf et al, 1997;Niwa et al, 1998). Growth of transfected BAF ± B03 pro-B cells needed the contribution of both signals derived from gp130: STATs induced anti-apoptotic factors, whereas MAP kinase activation was necessary for proliferation .…”