Serum regulation of Id1 expression by a BMP pathway and BMP responsive element

Immediate early genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP responsive element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad 4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.     

Autocrine regulation of terminal differentiation by interleukin-6 in the pluripotent KU812 cell line

The human KU812 leukemic cell line is a model for studying cell commitment towards different hematopoietic lineages. Indeed, this cell line is characterized by both a capacity for self-renewal and the ability to differentiate spontaneously along erythroid and basophilic cell lineages. In this study we show that interleukin-6 (IL-6) and its specific receptor (IL-6-R) are spontaneously expressed in the human KU812 cell line. Addition of antibody against IL-6 weakly inhibited its cell proliferation (20 to 30%) suggesting that the endogenous production of IL-6 was partially responsible for the growth of the cell line. In contrast, the spontaneous terminal differentiation of this cell line towards the erythroid and basophilic lineages was inhibited by an antibody against IL-6 and this effect was reversed by addition of recombinant human IL-6 (rIL-6). These results suggest that IL-6 is involved more in differentiation than in the proliferation of KU812 cells. After several passages, KU812 cells lose their capacity to differentiate spontaneously. In these cells, the IL-6-R was no more detectable. We therefore suggest that this loss of spontaneous differentiation is associated with an interruption of the IL-6 autocrine loop.     

Purification of an insulin-related factor secreted by a teratoma-derived mesodermal cell line

Insulin-related factor (IRF), a polypeptide secreted by the mouse teratoma-derived cell line (1246-3A), was purified 3210-fold to homogeneity from 1246-3A conditioned medium using a rapid three-step procedure including cation-exchange chromatography, immunoaffinity chromatography using a monoclonal antibody against porcine insulin coupled to an agarose gel support, and reverse phase high performance liquid chromatography. 10 micrograms of IRF was purified from 6 liters of 1246-3A conditioned medium. Pure IRF appeared as a single band with the same mobility as insulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRF stimulates cell proliferation of insulin-dependent cell line 1246 and competes with 125I-insulin for binding to 1246 cells; half-maximal growth stimulation and binding competition were achieved at an IRF concentration of 6.5 ng/ml (1.3 nM) and 25 ng/ml (4 nM), respectively, comparable with those for bovine insulin. The biochemical, biological, and immunological characteristics of IRF, as well as its amino acid composition, strongly suggest that it is closely related to pancreatic insulin in structure and function.     

BMP signaling and stem cell regulation

Stem cells play an essential role in cellular specialization and pattern formation during embryogenesis and in tissue regeneration in adults. This is mainly due to a stem cell's ability to replenish itself (self-renewal) and, at the same time, produce differentiated progeny. Realization of these special stem cell features has changed the prospective of the field. However, regulation of stem cell self-renewal and maintenance of its potentiality require a complicated regulatory network of both extracellular cues and intrinsic programs. Understanding how signaling regulates stem cell behavior will shed light on the molecular mechanisms underlying stem cell self-renewal. In this review, we focus on comparing the progress of recent research regarding the roles of the BMP signaling pathway in different stem cell systems, including embryonic stem cells, germline stem cells, hematopoietic stem cells, and intestinal stem cells. We hope this comparison, together with a brief look at other signaling pathways, will bring a more balanced view of BMP signaling in regulation of stem cell properties, and further point to a general principle that self-renewal of stem cells may require a combination of maintenance of proliferation potential, inhibition of apoptosis, and blocking of differentiation.     

The morphology and growth of a pluripotent teratocarcinoma cell line and its derivatives in tissue culture

Cultures of the clonally derived pluripotent teratocarcinoma cell line, SIKR, are heterogeneous. They are characterized by the presence of two cell types—the “C cells”, which grow as tight, round colonies on a monolayer of the morphologically distinct “E cells”. In contrast to the C cells, whose proliferation is apparently uninhibited by high cell density, the E cells show density-dependent inhibition of growth. Subclones of SIKR are of two types: they are either similar to the parent culture, in that they contain both C and E cells (CE subclones) and are themselves tumorigenic and pluripotent; or they are composed only of E cells (E-type subclones), which are primarily not tumorigenic, but may become so after spontaneous transformation in vitro. The tumors formed by transformed E cells (E-t cells) are monotypic (“fibroblastic”), consisting of one cell type which is not clearly identifiable, but which is distinctly not embryonal carcinoma.It is concluded that the tumorigenic C cells are the stem cells of this teratocarcinoma line, and that they give rise to nontumorigenic E cells in vitro, but that the reverse does not occur. It is suggested that the C to E transition represents cell determination in vitro. The interest of this cell culture system for both developmental and oncological studies is discussed.     

The BMP antagonist Noggin promotes cranial and spinal neurulation by distinct mechanisms

Here we characterize the consequences of elevated bone morphogenetic protein (BMP) signaling on neural tube morphogenesis by analyzing mice lacking the BMP antagonist, Noggin. Noggin is expressed dorsally in the closing neural folds and ventrally in the notochord and somites. All Noggin-/- pups are born with lumbar spina bifida; depending on genetic background, they may also have exencephaly. The exencephaly is due to a primary failure of neurulation, resulting from a lack of mid/hindbrain dorsolateral hinge point (DLHP) formation. Thus, as previously shown for Shh signaling at spinal levels, BMP activity may inhibit cranial DLHP morphogenesis. However, the increased BMP signaling observed in the Noggin-/- dorsal neural tube is not sufficient to cause exencephaly; it appears to also depend on the action of a genetic modifier, which may act to increase dorsal Shh signaling. The spinal neural tube defect results from a different mechanism: increased BMP signaling in the mesoderm between the limb buds leads to abnormal somite differentiation and axial skeletal malformation. The resulting lack of mechanical support for the neural tube causes spina bifida. We show that this defect is due to elevated BMP4 signaling. Thus, Noggin is required for mammalian neurulation in two contexts, dependent on position along the rostrocaudal axis.     

Differential regulation of Dlx gene expression by a BMP morphogenetic gradient.

Three members of the vertebrate Distal-less gene family, Dlx3, 5 and 6, are transcribed in early gastrula embryos of Xenopus laevis. This expression is confined to ectoderm and is excluded from the presumptive neural plate region. Expression of all three genes is dependent upon BMP signaling, with significant differences in how the three genes respond to the BMP antagonist chordin. This correlates with the different expression domain boundaries in vivo for Dlx3 compared to Dlx5 and 6, suggesting that BMP signal attenuation could be the primary factor in determining these different patterns in the gastrula ectoderm.     

Noggin1 and Follistatin-like2 function redundantly to Chordin to antagonize BMP activity

In Xenopus, the dorso-ventral (D/V) axis is thought to be specified by the bone morphogenetic proteins (Bmp) activity arising through interaction with antagonists such as Noggin, Chordin and Follistatin. We report here, through inactivation of noggin1 (nog1) that this gene is not essential by itself to establish the D/V patterning. However, at blastula stage, inactivation of nog1 strongly amplifies chordin (chd) phenotype, revealing redundant functions of these two genes on D/V axis formation. Substantial dorsal tissues remaining in the double nog1-chd morphant suggested that other anti-Bmp factors may pattern the D/V axis. We isolated two potential candidates, the follistatin-like (fstl) genes. We found that fstl2 is an early gastrula expressed gene. Its inactivation, similar to nog1, strongly enhances the chd phenotype. Moreover, the penetrance of the ventralization phenotype is much higher when we inactivated simultaneously chd, nog1 and fstl2. Altogether, our data reveal that, while Chordin is the main player of the D/V axis, sufficient to maintain proper activity of Bmp gradient, the structures remaining in the chd mutant (namely dorsal and dorso-lateral territories, in both mesodermal and ectodermal layers) result from the anti-Bmp activity carried by Nog1 and Fstl2 at blastula and gastrula stages.     

Osteogenic protein-1 (OP-1, BMP-7) induces osteoblastic cell differentiation of the pluripotent mesenchymal cell line C2C12

The effects of Osteogenic Protein-1 (OP-1, BMP-7) on the differentiation of the pluripotent mesenchymal cell line, C2C12, were examined. OP-1 at 50 ng/ml partially inhibited myotube formation in C2C12 cells, while OP-1 at 200 ng/ml completely inhibited myotube formation and induced the formation of cells displaying osteoblastic morphology. High concentrations of OP-1 elevated the alkaline phosphatase (AP) activity dramatically, both as a function of time and OP-1 concentration. Osteocalcin (OC) mRNA expression was detected as early as 8 days in OP-1-treated cultures and subsequently increased considerably. Expression of bone sialoprotein (BSP) mRNA was low in control cultures and stimulated by OP-1. Collagen type I mRNA expression was enhanced by OP-1 during the early days in culture, but gradually decreased thereafter. MyoD mRNA expression, high in control cultures, was suppressed by OP-1 in a dose- and time-dependent manner. OP-1 enhanced ActR-I mRNA expression and significantly elevated the mRNA expressions of BMP-1, BMP-4, BMP-5, GDF-6, and GDF-8. The present results indicate that OP-1 is a potent inducer of C2C12 differentiation into osteoblastic cells.     

Epigenetic regulation of Nanog expression by Ezh2 in pluripotent stem cells

Nanog levels in pluripotent stem cells are heterogeneous and this is thought to reflect two different and interchangeable cell states, respectively poised to self-renew (Nanog-high subpopulation) or to differentiate (Nanog-low subpopulation). However, little is known about the mechanisms responsible for this pattern of Nanog expression. Here, we have examined the impact of the histone methyltransferase Ezh2 on pluripotent stem cells and on Nanog expression. Interestingly, induced pluripotent stem (iPS) cells lacking Ezh2 presented higher levels of Nanog due to a relative expansion of the Nanog-high subpopulation, and this was associated to severe defects in differentiation. Moreover, we found that the Nanog promoter in embryonic stem (ES) cells and iPS cells coexists in two alternative univalent chromatin configurations, either H3K4me3 or H3K27me3, the latter being dependent on the presence of functional Ezh2. Finally, the levels of expression of Ezh2, as well as the amount of H3K27me3 present at the Nanog promoter, were higher in the Nanog-low subpopulation of ES/iPS cells. Together, these data indicate that Ezh2 directly regulates the epigenetic status of the Nanog promoter affecting the balance of Nanog expression in pluripotent stem cells and, therefore, the equilibrium between self-renewal and differentiation.     

Establishment of a pluripotent embryonal carcinoma cell line not expressing SSEA-1 and ECMA-7 phenotypes

A murine embryonal carcinoma (EC) cell line heterozygous for t0 recessive lethal mutation has been established from an embryo-derived transplantable teratocarcinoma TC1Ph of the genotype (129-T/t0 X C3H/Di)t0/+. The EC cell line, designated EC1Ph, and two cloned sublines, EC1Ph/a and EC1Ph/b, maintain the diploid karyotype (40, XY) and give rise to teratocarcinomas with differentiated derivatives of EC cells after inoculation into syngeneic recipients. The cloned sublines express low or zero amounts of SSEA-1 and ECMA-7 stage-specific antigens. At some passages, the EC1Ph line and the cloned subline EC1Ph/b express a significant quantity of class I H-2 antigens. This unusual EC phenotype resembles that of human teratocarcinoma cell lines.     

A T lymphoid cell line responds to a thymic stromal cell line by expression of Thy-1 and CD4

We have cloned both T lymphoid and stromal lines from a single murine thymic tumor that was induced by a retrovirus carrying the v-myc oncogene (M-MuLV(myc]. The T lymphoid line, L4, was cloned by growth in agar. L4 cells were initially negative for Thy-1.2 and CD4 (although they contained rearranged TCR-beta genes), and they remained so if passaged in medium alone. However, cocultivation of these Thy-1.2- CD4- cells with the cloned stromal cell line, St3, resulted in sequential expression of Thy-1.2 and CD4 in subpopulations of cells. Thy-1.2+ CD4- and Thy-1.2+ CD4+ L4 subclones were obtained from the cocultures by subsequent cloning in agar. Derivation of these subclones from the starting Thy-1.2- CD4- clone was verified by Southern blot analyses specific for TCR-beta gene rearrangements and for M-MuLV(myc) proviral integration sites. Continuous cocultivation of Thy-1.2+ CD4+ L4 subclones with the St3 stromal cells was necessary for maintenance of CD4 on the cell surface. Furthermore, CD4 expression which was lost when CD4+ L4 cells were removed from the stroma could be reinduced if they were again cultured on St3 stroma. These cells may provide a model system for studying thymocyte-stromal cell interactions in induction and maintenance of expression of Thy-1 and CD4 molecules.     

BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis

Esophageal atresia with tracheoesophageal fistula (EA/TEF) is a serious human birth defect, in which the esophagus ends before reaching the stomach, and is aberrantly connected with the trachea. Several mouse models of EA/TEF have recently demonstrated that proper dorsal/ventral (D/V) patterning of the primitive anterior foregut endoderm is essential for correct compartmentalization of the trachea and esophagus. Here we elucidate the pathogenic mechanisms underlying the EA/TEF that occurs in mice lacking the BMP antagonist Noggin, which display correct dorsal/ventral patterning. To clarify the mechanism of this malformation, we use spatiotemporal manipulation of Noggin and BMP receptor 1A conditional alleles during foregut development. Surprisingly, we find that the expression of Noggin in the compartmentalizing endoderm is not required to generate distinct tracheal and esophageal tubes. Instead, we show that Noggin and BMP signaling attenuation are required in the early notochord to correctly resolve notochord cells from the dorsal foregut endoderm, which in turn, appears to be a prerequisite for foregut compartmentalization. Collectively, our findings support an emerging model for a mechanism underlying EA/TEF in which impaired notochord resolution from the early endoderm causes the foregut to be hypo-cellular just prior to the critical period of compartmentalization. Our further characterizations suggest that Noggin may regulate a cell rearrangement process that involves reciprocal E-cadherin and Zeb1 expression in the resolving notochord cells.     

Altered regulation of P-450IA1 expression in a multidrug-resistant MCF-7 human breast cancer cell line

MCF-7 human breast cancer cells, selected for resistance to adriamycin (AdrR), exhibit the phenotype of multidrug resistance (MDR). Previous studies have shown that resistance in AdrR MCF-7 cells is associated with several biochemical changes that are similar to those induced in rat hyperplastic nodules, preneoplastic liver lesions which display broad spectrum resistance to carcinogens and hepatotoxins. In this report, we show that these changes in the AdrR MCF-7 cells are also associated with the development of cross-resistance to the procarcinogen benzo(a)pyrene (BP) and are associated with a marked defect in the conversion of BP to its cytotoxic, carcinogenic metabolites by AdrR cells. Since aryl hydrocarbon hydroxylase is the principle enzyme activity which converts benzo(a)pyrene to toxic hydroxylated forms, the regulation of cytochrome P-450IA1 expression, the gene encoding this enzyme activity in MCF-7 cells, was examined. Incubation with 100 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h results in a marked increase in aryl hydrocarbon hydroxylase activity in wild type (WT) but not AdrR MCF-7 cells. The alteration in aryl hydrocarbon hydroxylase expression in the AdrR cells is not overcome by incubation either with higher concentrations of TCDD (1 microM) or for longer periods of time (4 days). Northern blot analysis indicates that this defect in AdrR MCF-7 cells involves a regulatory defect at the level of P-450IA1 RNA. Following transfection of a construct containing the normal mouse P-450IA1 promoter fused to a reporter gene (bacterial chloramphenicol acetyltransferase) into WT and AdrR MCF-7 cells, TCDD induced chloramphenicol acetyltransferase activity in WT MCF-7 cells only. Furthermore, TCDD also induces both DT-diaphorase and UDP-glucuronyltransferase activities in WT, but not AdrR cells. These data suggest that the defect in the AdrR MCF-7 cells is not due to a structural P-450IA1 gene mutation, but rather involves a product regulating the polycyclic hydrocarbon-inducible expression of several drug-metabolizing enzyme activities. This defect in the AdrR MCF-7 cells is also associated with the development of resistance to ellipticine, an anticancer agent which is converted to more toxic hydroxylated species by aryl hydrocarbon hydroxylase or a similar mixed function oxidase. The WT and AdrR MCF-7 cells represent a useful model to study the regulation of the P-450IA1 gene in human cells.