Although a crucial role for menin in suppression of tumorigenesis is well documented, little is known about its biochemical function (27
). Menin interacts with a number of nuclear proteins, including JunD, NF-κb, Smad3, and Pem (6
) and represses the activities of some of these factors in reporter gene assays. Overexpression of menin suppresses insulin-induced transcription of c-Fos(10
). Ectopic expression of menin has also been reported to repress the promoters of prolactin and gastrin (28
), based on luciferase reporter assays. However, so far little is known about whether menin is essential for repression of endogenous genes. Using menin-null cells generated by gene-targeting, we show that menin is essential for repressing the expression of an endogenous gene, IGFBP-2 (-). Complementation of the menin-null cells with menin inhibits the expression of IGFBP-2. Moreover, a common menin mutation from MEN1 patients, K119Δ, fails to repress the IGFBP-2 expression, although the level of expression of K119Δ is even higher than that of wild type menin. In addition, mutations of each NLS1 and NLS2 in menin fail to block the nuclear translocation of menin but compromise the ability to repress IGFBP-2 expression, indicating an additional role of the NLSs in repressing IGFBP-2.
These findings have several important implications for understanding the role of menin in regulation of gene transcription. First, these studies indicate that menin is essential for repression of the endogenous IGFBP-2 gene, since targeted disruption of Men1
in MEFs leads to activation of the IGFBP-2, a gene involved in both positive and negative regulation of cell proliferation, depending on cell type (13
). Second, the menin-mediated repression of IGFBP-2 is at least in part executed through the promoter region (-). Third, the fact that a point mutant, K119Δ, fails to repress expression of IGFBP-2 strongly suggest that menin-mediated regulation of IGFBP-2 may be in part related to the function of menin in suppressing the development of MEN1. Finally, the fact that the NLS1 and 2 double mutant menin is still able to enter the nucleus indicates the existence of additional NLSs in menin. Currently it is not known where the additional NLS is located. Furthermore, the NLS1 and 2 are not only important for the nuclear localization, but also crucial for repressing the expression of IGFBP-2 (). These findings provide an opportunity to further investigate how menin regulates endogenous genes.
IGFBP-2 is a member of the IGF binding protein family (11
). It inhibits cell proliferation induced by IGFs (11
). In support of this function, transgenic mice overexpressing IGFBP-2 display significantly reduced body weight (11
). Consistent with this, IGFBP-2 mediates inhibition of cell proliferation that is induced by transforming growth factor-beta (TGF-ß) (14
). This is particularly interesting since TGF-ß usually inhibits proliferation of epithelial cells, but stimulates proliferation of cancer cells that harbor TGF-ß-resistant oncogenes (15
). On the other hand, IGFBP-2 also possesses IGF-independent activity. For example, expression of IGFBP-2 increases the tumorigenesis of adrenal cortical cancer cells (12
). In addition, IGFBP-2 also stimulates the growth of prostate cancer cells (13
) and the metastasis of glioblastoma invasion (30
). Furthermore, it is also a highly expressed marker for many cancers including prostate cancer and colon cancer (11
). It is still unclear how exactly IGFBP-2 induces tumorigenesis.
In the current study, we fail to observe IGFBP-2-induced cell proliferation in the menin-null MEFs. Several explanations may exist for this observation. First, it is possible that the MEFs may not be a cell type in which IGFBP-2 induces cell proliferation. Second, IGFBP-2 may act similar to TGF-β, and inhibit proliferation of normal cells but stimulate proliferation only in cancer cells that harbor certain types of oncogenes, which are not present in the MEFs used in these studies Third, it is also formally possible that IGFBP-2 is not the major target gene that is crucial for menin-induced inhibition of cell proliferation. However, studying menin-induced expression of IGFBP-2 will provide novel insights into how menin regulates gene expression.
It is unclear how menin represses the expression of IGFBP-2. We previously showed that a majority of menin closely associates with chromatin (20
). Here we show that menin inhibits the promoter of IGFBP-2 in a dose-dependent manner (), and a common mutation from MEN1 patients fails to inhibit the promoter of IGFBP-2 (). In addition, menin also alters the status of the chromatin structure surrounding the IGFBP-2 promoter (). It has recently been shown that menin inhibits the expression of telomerase (hTERT), by binding to the putative AP1 and NF-κb binding sites in the promoter of hTERT (31
). Menin could inhibit these transcription factors by recruiting mSin3A and histone deacetylase (32
). Menin is also recently shown to associate with a histone methyltransferase complex and activate transcription of the endogenous Hoxc-8 gene (34
). Thus, it is possible that menin interacts with other co-regulators such as the mSin3A-histone deacetylase complex to repress the expression of IGFBP-2, at least in part, through the promoter containing multiple Sp1 binding sites.
Further supporting the role of menin in regulating IGFBP-2, NLS1 and NLS2 in menin are each crucial for repressing the IGFBP-2 expression (), although deletion of NLS1 or NLS2 alone does not block the nuclear translocation of menin. These results imply that these NLSs are essential for repressing IGFBP-2 in addition to targeting menin to the nucleus. Perhaps the positively charged NLSs may mediate menin’s association with other factors or nuclear structures that modulate transcription. Although the detailed mechanism for repression of IGFBP-2 by menin remains to be determined, the current studies have established that menin is essential for optimal repression of IGFBP-2, a protein that plays crucial roles in positive and negative regulation of cell proliferation.