DPP homologs and programmed cell death

The mechanisms by which programmed cell death is spatially regulated are not well characterized. Msx1 and Msx2 (Homologs of Drosophila Muscle segment homeobox) are two closely related homeobox-containing genes that are expressed at sites where cellular proliferation and programmed cell death occur, including the developing limb and the cephalic neural crest. Tissue interactions are necessary for the maintenance of Msx1 and Msx2 expression and programmed cell death. It has been demonstrated that BMP4 can regulate cell death at these same sites as well as induce Msx expression. These observations lead to the hypothesis that Msx2 is a key regulator of cell death in the BMP-mediated pathway. Embryonic stem (ES) cell lines will undergo processes typical of early embryogenesis upon aggregation and have recently been shown to provide a model system for programmed cell death. In contrast to ES cells, P19 cells do not undergo pronounced cell death upon aggregation; however, constitutive ectopic Msx2 expression in P19 cells results in a marked increase in apoptosis induced upon aggregation but has no effect when cells are grown as a monolayer. If aggregates are allowed to interact with a substrate, the process of programmed cell death is completely inhibited. Addition of BMP4 to aggregated P19 cells also results in cell death; however, BMP4 does not increase levels of cell death in Msx2-expressing cells. Addition of BMP4 to P19 cells results in an induction of Msx2 transcription consistent with Msx2's proposed role in cell death in the embryo. These data support a model by which BMP4 induces programmed cell death via an Msx2-mediated pathway and provide direct functional evidence that Msx2 expression is a regulator of this process (Marazzi, 1997).

Neuron numbers in developing vertebrate organisms are regulated by the availability of growth factors that promote neuron survival. However, neuron survival may also be regulated by growth factors that promote rather than prevent cell death. This study examines the effects of bone morphogenetic proteins (BMPs) in inducing apoptosis in MAH cells, a line of immortalized sympathoadrenal progenitor cells. Treatment of MAH cells with BMP2 or BMP4 kills the cells in a dose-dependent manner. In contrast to this, treatment with BMP7 or TGFbeta1 fails to affect survival, suggesting that induction of apoptosis is specific to the Dpp subgroup of BMPs. Survival after treatment with BMP2 or BMP4 requires addition of fibroblast growth factor (FGF) and nerve growth factor (NGF), indicating that BMP treatment makes the neurons dependent on an exogenous factor for survival. Several experimental observations suggest an apoptotic mechanism for BMP-induced death. After BMP2 treatment, the cells progressively shrink and become pyknotic. There is prominent endonucleosomic cleavage of DNA (laddering) as well as TUNEL staining. BMP-induced death is inhibited by the caspase inhibitor z-VAD and is partially prevented by the endonuclease inhibitor aurintricarboxylic acid. These observations suggest that neuron numbers may be regulated by factors that promote death and that exposure to such factors may be a signal for the development of dependence on other growth factors for survival (Song, 1998).

The effects of Bmp-4 on interdigital cell death were investigated in the mouse. Affi-Gel beads, loaded with recombinant Bmp-4 protein, were transplanted into the interdigital tissues of day 12.5 hindlimb, ex utero. It was established that Bmp-4 can induce precocious interdigital cell death. Using in situ hybridization, the expression patterns of bmp-4 and alk-6 receptor were established. Both genes are coexpressed in the interdigital region of 12.5- and 13.5-day hindlimbs. This suggests that Bmp-4 may act in an autocrine fashion. The effects of Bmp-4 on 12.5-day interdigital tissue cultures was examined. In all specimens examined, the interdigital tissues produce cartilage instead of participating in cell death. The addition of exogenous Bmp-4 to the interdigital cultures does not induce apoptosis but instead enhances chondrogenesis. The discrepancy between the effects of Bmp-4 in vitro and ex utero is attributed to the presence of digits. When a flanking digit is left attached to the interdigital tissues, in vitro, Bmp-4 promotes apoptosis instead of chondrogenesis. In sum, the results suggest that Bmp-4 is a multifunctional protein and its effect on the interdigital tissues is dependent on the modulating influence of the digits (Tang, 2000).

The role of FGFs in the control of programmed cell death during limb development has been investigated by analyzing the effects of increasing and blocking FGF signaling in the avian limb bud. BMPs are currently considered to be the signals responsible for cell death. FGF signaling is also necessary for apoptosis and the establishment of the areas of cell death is regulated by the convergence of FGF- and BMP-mediated signaling pathways. Cell death is inhibited for short intervals (12 hours) after administration of FGFs. However, this initial inhibition is followed (24 hours) by a dramatic increase in cell death, which can be abolished by treatments with a BMP antagonist (Noggin or Gremlin). Conversely, blockage of FGF signaling by applying a specific FGF-inhibitor (SU5402) into the interdigital regions inhibits both physiological cell death and cell death mediated by exogenous BMPs. Furthermore, FGF receptors 1, 2 and 3 are expressed in the autopodial mesoderm during the regression of the interdigital tissue, and the expression of FGFR3 in the interdigital regions is regulated by FGFs and BMPs in the same fashion as apopotosis. Together these findings indicate that, in the absence of FGF signaling BMPs are not sufficient to trigger apoptosis in the developing limb. Although evidence is provided for a positive influence of FGFs on BMP gene expression, the physiological implication of FGFs in apoptosis appears to result from their requirement for the expression of genes of the apoptotic cascade. MSX2 and Snail have been identified as candidate genes associated with apoptosis and their expression requires the combined action of FGFs and BMPs (Montero, 2001).

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