As in a previous study in choriocarcinoma cells, leptin treatment did not activate STAT3 phosphorylation in primary mouse trophoblast cells [17
]. However, phosphorylated STAT3 was detectable in the treated and untreated cells, a result that contrasts with findings in BeWo cells, which displayed no phosphorylated STAT3 either before or after leptin treatment [17
]. Although leptin treatment did not alter the status of STAT3 phosphorylation, it did increase the expression of SOCS3. The rapid increase in SOCS3 protein (within 1 h) might be due to an increase in the stability of the protein, as has been demonstrated for SOCS3 in other systems [31
]. However, SOCS3 mRNA induction has been detected in response to leptin in as little as 30 min in rat INS-1 insulinoma cells [32
]. Therefore, the increase in SOCS3 could be an outcome of transcriptional upregulation of its gene, increased mRNA stability, reduced protein turnover, or a combination of these three factors. SOCS3 has been identified as a negative feedback regulator of activated STAT3 signaling by the leptin receptor and functions to inhibit JAK2 activation [33
]. Our finding of SOCS3 activation in the absence of any change in STAT3 phosphorylation status suggests that SOCS3 is acting in an independent signaling capacity and not as part of a STAT3 feedback loop. A somewhat similar effect occurs in pancreatic beta cells, in which leptin treatment induces both STAT3 and SOCS3 activity. However, the net effect of leptin treatment on gene expression reflected the direct action of SOCS3 rather than its ability to antagonize STAT3 [32
SOCS3 induction is generally considered to occur as a direct outcome of STAT3 phosphorylation, as has been observed in pancreatic beta cells [32
] and some other systems [34
]. In the RCHO-1 immortalized rat trophoblast cell line, expression of SOCS3 is both STAT3 and MAP2K1 (MEK) dependent [36
]. Because both are present but only MEK is being activated by leptin treatment, it is likely that constitutively phosphorylated STAT3 and leptin-stimulated MAP2K1 (MEK) action are driving the increased SOCS3 expression in our experiments.
Our finding that MAP2K1 (MEK) shows an immediate increase in phosphorylation after leptin treatment of trophoblast cells is consistent with the activation of MAPK3/MAPK1 (ERK 1/2) by leptin in BeWo and JAr choriocarcinoma cells [17
]. Thus, even though leptin stimulates proliferation in choriocarcinoma cells and appears not to do so in primary trophoblasts, there is an apparent conservation of this cell signaling mechanism [17
]. As in primary human trophoblast cultures [10
], leptin promoted a modest increase in MMP activity in the mouse cells, which likely contributes to their increased invasive potential. In the presence of the MAP2K1 (MEK) inhibitor U0126, leptin was unable to stimulate MMP activity. However, the stimulatory effect of the inhibitor vehicle, DMSO, masked much of the leptin effect. Thus, although the inhibitor data are consistent with a role for MAP2K1 (MEK) in mediating leptin-stimulated MMP activity, we are unable to reach an unequivocal conclusion.
STAT3 is highly expressed in cultured human trophoblasts retrieved early in pregnancy, when extravillous trophoblast cells are highly invasive. Inhibition of STAT3 in such cells is effective at reducing invasiveness [37
]. In mice, differentiation of invasive giant cells is regulated by the balance between LIF signaling via STAT3 and SOCS3 [39
]. Although the overall effect of leptin is to promote trophoblast invasion in vitro [19
], in the present study it inhibited STAT3. Indeed, the STAT3 inhibitor cucurbitacin I significantly inhibited MMP activity only in the presence of leptin, consistent with a further suppression of STAT3 activity. Thus, leptin may have a mixed effect on trophoblast invasion, with simultaneous activation of MAP2K1 (MEK) signaling and inhibition of STAT3 signaling. It will be interesting to determine the net effect of leptin in the presence of factors such as LIF that activate trophoblast invasion via the STAT3 pathway [14
The microarray data provide general support for the conclusion that MAP2K1 (MEK) signaling is being influenced by leptin treatment. For example, the “MAPK cascade” was identified as a significantly represented functional category in all three gene lists in the DAVID analysis, although it was not among the highest-scoring categories, which are listed in . The gene for MEK itself (Map2k1) was expressed significantly higher at 24 h in the leptin-treated versus control samples.
Not surprisingly, the MEK pathway was only one of many signaling pathways affected by leptin treatment and/or by time in culture. There were changes in gene expression of several members of the RhoGTPase family in all three comparisons, including increases over time in expression of Rhoc
(RhoE) in leptin-treated but not control cells. In addition, extremely small (less than 1.5-fold) but statistically significant differences in Rhoa
(higher in leptin) and Rock2
(higher in control) were seen at 1 h, suggesting dynamic regulation of this pathway by leptin, but not a specific direction of change. Both RHOA and RHOC are involved in control of focal adhesion and cell motility in many cell types, including human extravillous trophoblasts [41
]. In addition, both MAP2K1 (MEK) and rho-dependent kinase activity have been implicated in leptin stimulation of invasion in cancer cells [42
]. In pancreatic tumor cells, RHOC promotes invasion following loss of caveolin 1 [43
]. The observation that the gene for caveolin 1 (Cav1
) is significantly downregulated at 6 h in our experiments is consistent with a role for RHOC in directing leptin-stimulated invasion of mouse trophoblast cells.
A comparison of gene expression profiles at 1 h and 6 h after leptin addition to the cultures indicates a general downregulation of both proapoptotic and antiapoptotic genes. For example, there is lowered expression of antiapoptotic Bcl2-related genes (Bcl2
, and Bcl2a1d
), as well as proapoptotic granzymes (Gzm d
, and g
) and caspases (Casp 4
), but these changes may be unrelated to the action of leptin. Rather, they are probably correlated with cell death occurring during the first hours of culture, because by 24 h, differences in expression of the majority of apoptotic genes between leptin and control cultures were not evident (d and Supplemental Data), and the apoptosis pathway was not on the list of significantly enriched functional categories for genes regulated significantly by leptin over 24 h (). Interestingly, some of the granzyme genes (Gzm d
, and g
) and Casp4
had significantly higher expression in the leptin-treated cells at 24 h. We are unable to explain these late changes, although it is possible that the granzymes are serving a function unrelated to apoptosis, such as in digesting extracellular matrix components [44
The primary goal of this paper was to identify intracellular mechanisms whereby leptin increases trophoblast invasion. Accordingly, among the functional categories of genes regulated by leptin on the microarrays, “cell motility” (enrichment score 0.37; 19 genes for time-leptin interaction) is of particular interest. The most upregulated gene in this grouping was Stmn1
, whose changed expression was confirmed by real-time PCR analysis. Its product, stathmin, has been identified as a key effecter of cell migration in Drosophila
border cells, a model of normal physiological (nonmetastatic) cell migration [45
]. Stathmin also promotes invasiveness of sarcoma cells [46
] and motility of cultured gonadotropin-releasing hormone neurons [47
]. The protein is a well-established regulator of microtubule dynamics during mitotic spindle formation, and indeed it was also classified in the “cell cycle” and “mitotic spindle” categories by the cluster analysis program. Its role as a regulator of microtubule dynamics is likely responsible for its involvement in the category “control of cell motility” [48
]. Thus, stathmin is a good candidate for further investigations of mechanisms of trophoblast invasion, and particularly of how leptin stimulates this and related processes in other cell types.
Stathmin may play a second role in invading trophoblast cells; namely, in controlling endoduplication, because stathmin downregulation is required for genome duplication and endopolyploidy in megakaryocytes [49
]. Similarly, geminin (Gmnn
), whose expression was maintained by the presence of leptin over 24 h, is repressed during megakaryocyte differentiation [50
]. Moreover, deletion of Gmnn
induces endoreduplication and premature differentiation of giant cells in the early mouse embryo [51
]. In contrast, Aurka
(aurora kinase a), which was downregulated by leptin over 24 h, is positively associated with polyploidy [52
]. Similarly, MacAuley et al. [54
] reported a switch from cyclin D3 to D1 with the onset of endoreduplication in mouse giant cells, whereas we observed a decrease in Ccnd1
expression with leptin treatment relative to controls at 24 h. Together, these data are best explained if leptin treatment is inhibiting rather than promoting genome endoreduplication in these primary trophoblast cells. At first glance, however, the Feulgen data appear to contradict this finding. indicates that the leptin-treated cells had a higher average DNA content than the control groups. On the other hand, careful examination of the frequency distribution of cells over the range of DNA contents revealed a more subtle effect. After leptin treatment there was a shift in the curve from cells of low DNA content to ones with a midrange content, but little change in the number of cells with the highest amounts of DNA. In other words, there may be no increase in the number of terminally differentiated giant cells, merely a shift toward cells of intermediary phenotype.
Parast et al. [55
] observed three stages of giant cell differentiation in Rcho-1 and primary rat trophoblast cultures. In the earliest stage, cells were nonmotile and proliferative. In the second, cells were highly motile but not dividing. In the third, terminally differentiated stage, cells were nonmotile and nonproliferative, with well-developed cell junctions. A similar phenomenon likely occurs in mouse trophoblast cells. Our data suggest that leptin is driving stage 1 cells to stage 2 and perhaps inhibiting the transition to stage 3. In particular, the Feulgen staining indicates a loss of cells at the earliest stage of differentiation and no gain in stage 3, whereas the array data are consistent with the view that leptin slows terminal differentiation. One possible explanation for the above results is that leptin somehow modulates transforming growth factor beta (TGFB) signaling, which has been implicated in driving terminal differentiation of trophoblasts of Rcho cells [56
] and inhibiting invasion of primary and immortalized human trophoblast cells [58
]. That leptin might promote early differentiation events but inhibit advancement to a terminally differentiated state is supported by the observation that genes associated with the TGFB signaling pathway are initially upregulated by leptin () but subsequently show decreased expression. In particular, transcript concentrations for TGFBR2 and SMAD3 decline by 24 h, whereas those representing the inhibitor decorin are increased relative to controls.
It will be important to determine how these in vitro differentiation stages correlate with in vivo differentiation events and with the subpopulations of trophoblast giant cells that are present in the mature mouse placenta. Giant cells, as defined by their elevated ploidy, are found in multiple locations in the placenta and may have quite different phenotypes and functions [60
]. To better understand how leptin may be affecting the differentiation of precursor cells to these subtypes, we examined regulation of genes identified as being preferentially expressed in one or more subpopulations of giant cells in vivo [60
]. At 24 h, leptin upregulated genes said to specifically mark several different subtypes: Ctsq
(cathepsin q), Prl3d1
(prolactin family 3, subfamily d, member 1, or placental lactogen 1), Prl2a1
, and Prl7d1
(prolactin family 7, subfamily d, member 1, or proliferin-related protein; and Supplemental Data). Thus, there is no evidence that leptin drives differentiation of any one of these in vivo subtypes at the expense of the others, or that any of these marker subtypes individually represents the “stage 2” differentiation that we see in vitro.
Overall, our data indicate that leptin acts through several signal transduction intermediates, including MAP2K1 (MEK) and SOCS3, to influence the differentiation of primary trophoblast cells. We suggest that leptin accelerates disappearance of non-giant cells while inhibiting terminal differentiation of committed giant cells. Leptin's ability to maintain trophoblast cells at an intermediary stage of differentiation likely contributes to their increased invasiveness relative to controls.