BMP Responses Research Articles

Quantitative analysis of signaling responses during mouse primordial germ cell specification.

ABSTRACTDuring early mammalian development, the pluripotent cells of the embryo are exposed to a combination of signals that drive exit from pluripotency and germ layer differentiation. At the same time, a small population of pluripotent cells give rise to the primordial germ cells (PGCs), the precursors of the sperm and egg, which pass on heritable genetic information to the next generation. Despite the importance of PGCs, it remains unclear how they are first segregated from the soma, and if this involves distinct responses to their signaling environment. To investigate this question, we mapped BMP, MAPK and WNT signaling responses over time in PGCs and their surrounding niche in vitro and in vivo at single-cell resolution. We showed that, in the mouse embryo, early PGCs exhibit lower BMP and MAPK responses compared to neighboring extraembryonic mesoderm cells, suggesting the emergence of distinct signaling regulatory mechanisms in the germline versus soma. In contrast, PGCs and somatic cells responded comparably to WNT, indicating that this signal alone is not sufficient to promote somatic differentiation. Finally, we investigated the requirement of a BMP response for these cell fate decisions. We found that cell lines with a mutation in the BMP receptor (Bmpr1a−/−), which exhibit an impaired BMP signaling response, can efficiently generate PGC-like cells revealing that canonical BMP signaling is not cell autonomously required to direct PGC-like differentiation.

Sequence environment of BMP-dependent activating elements controls transcriptional responses to Dpp signaling in Drosophila.

Intercellular signaling pathways activate transcription factors, which, along with tissue-specific co-factors, regulate expression of target genes. Responses to TGFβ/BMP signals are mediated by Smad proteins, which form complexes and accumulate in the nucleus to directly bind and regulate enhancers of BMP targets upon signaling. In Drosophila, gene activation by BMP signaling often requires, in addition to direct input by Smads, the signal-dependent removal of the transcriptional repressor Brk. Previous studies on enhancers of BMP-activated genes have defined a BMP-responsive motif, the AE, which integrates activatory and repressive input by the Smad complex and Brk, respectively. Here, we address whether sequence variations within the core AE sequences might endow the motif with additional properties accounting for qualitative and quantitative differences in BMP responses, including tissue specificity of transcriptional activation and differential sensitivity to Smad and Brk inputs. By analyzing and cross-comparing three distinct BMP-responsive enhancers from the genes wit and Dad in two different epithelia, the wing imaginal disc and the follicular epithelium, we demonstrate that differences in the AEs contribute neither to the observed tissue-restriction of BMP responses nor to differences in the utilization of the Smad and Brk branches for transcriptional activation. Rather, our results suggest that the cis-environment of the BMP-response elements not only dictates tissue specificity but also differential sensitivity to the two BMP mediators.

Peak BMP Responses in the Drosophila Embryo Are Dependent on the Activation of Integrin Signaling.

SummaryWithin a 3D tissue, cells need to integrate signals from growth factors, such as BMPs, and the extracellular matrix (ECM) to coordinate growth and differentiation. Here, we use the Drosophila embryo as a model to investigate how BMP responses are influenced by a cell’s local ECM environment. We show that integrins, which are ECM receptors, are absolutely required for peak BMP signaling. This stimulatory effect of integrins requires their intracellular signaling function, which is activated by the ECM protein collagen IV. Mechanistically, integrins interact with the BMP receptor and stimulate phosphorylation of the downstream Mad transcription factor. The BMP-pathway-enhancing function of integrins is independent of focal adhesion kinase, but it requires conserved NPXY motifs in the β-integrin cytoplasmic tail. Furthermore, we show that an α-integrin subunit is a BMP target gene, identifying positive feedback between integrin signaling and BMP pathway activity that may contribute to robust cell fate decisions.

Id proteins are critical downstream effectors of BMP signaling in human pulmonary arterial smooth muscle cells

Bone morphogenetic protein type II receptor (BMPR-II) mutations are responsible for over 70% of cases of heritable pulmonary arterial hypertension (PAH). Loss of BMP signaling promotes pulmonary vascular remodeling via modulation of pulmonary artery smooth muscle cell (PASMC) proliferation. Id proteins (Id1-4) are major downstream transcriptional targets of BMP signaling. However, the impact of BMPR-II mutation on the expression of the range of Id proteins and the contribution of individual Id proteins to abnormal PASMC function remain unclear. Human PASMCs were used to determine the expression of Id proteins (Id1-4) by real-time PCR and immunoblotting. The BMP responses in control cells were compared with PASMCs harboring BMPR-II mutations and cells in which BMPR-II was knocked down by siRNA transfection. Id3 expression in pulmonary vessels was also investigated in BMPR-II mutant mice and in patients with heritable PAH. BMP4 and BMP6, but not BMP9, induced mRNA expression of Id1, Id2, and Id3. The BMP-stimulated induction of Id1 and Id3 was markedly reduced in BMPR-II mutant PASMCs and in control PASMCs following siRNA silencing of BMPR-II. Pulmonary arteries in BMPR-II mutant mice and patients with heritable PAH demonstrated reduced levels of Id3 compared with control subjects. Lentiviral overexpression of Id3 reduced cell cycle progression and inhibited proliferation of PASMCs. Lipopolysaccharide further reduced Id3 expression in mutant PASMCs. In conclusion, Id proteins, and particularly Id1 and Id3, are critical downstream effectors of BMP signaling in PASMCs. Loss of BMPR-II function reduces the induction of Id genes in PASMCs, Id1, and Id3 regulate the proliferation of PASMCs via cell cycle inhibition, an effect that may be exacerbated by inflammatory stimuli.

USP15 is a deubiquitylating enzyme for receptor-activated SMADs

The TGFβ pathway is critical for embryonic development and adult tissue homeostasis. On ligand stimulation, TGFβ and BMP receptors phosphorylate receptor-activated SMADs (R-SMADs), which then associate with SMAD4 to form a transcriptional complex that regulates gene expression through specific DNA recognition. Several ubiquitin ligases serve as inhibitors of R-SMADs, yet no deubiquitylating enzyme (DUB) for these molecules has so far been identified. This has left unexplored the possibility that ubiquitylation of R-SMADs is reversible and engaged in regulating SMAD function, in addition to degradation. Here we identify USP15 as a DUB for R-SMADs. USP15 is required for TGFβ and BMP responses in mammalian cells and Xenopus embryos. At the biochemical level, USP15 primarily opposes R-SMAD monoubiquitylation, which targets the DNA-binding domains of R-SMADs and prevents promoter recognition. As such, USP15 is critical for the occupancy of endogenous target promoters by the SMAD complex. These data identify an additional layer of control by which the ubiquitin system regulates TGFβ biology.

Expression and functional analysis of endoglin in isolated liver cells and its involvement in fibrogenic Smad signalling

Endoglin is an accessory component of the TGF-β-binding receptor complex that differentially modulates TGF-β and BMP responses. The existence of two splice variants L- and S-endoglin which differ in their cytoplasmic domain has already been shown in human and mice. Endoglin is located on the cell surfaces of cultured hepatic stellate cells and transdifferentiated myofibroblasts suggesting that this receptor might be associated with the profibrogenic attributes of these liver cell subpopulations. We now show that endoglin expression is increased in transdifferentiating hepatic stellate cells and in two models of liver fibrosis (i.e. bile duct ligation and carbon tetrachloride model) and further detectable in cultured portal fibroblasts representing another important fibrogenic cell type but not in hepatocytes. In respect to TGF-β1-signalling, we demonstrate that endoglin interacts with and is phosphorylated by TβRII. In hepatic stellate cells, TGF-β1 upregulates endoglin expression most likely via the ALK5 pathway and requires the SP1 transcription factor. We further identified a novel rat splice variant that is structurally and functionally different from that identified in human and mouse. Transient overexpression of endoglin resulted in a strong increase of TGF-β1-driven Smad1/5 phosphorylation and α-smooth muscle actin expression in a hepatic stellate cell line. In supernatants of respective cultures, we could detect the ectodomain of endoglin suggesting that shedding is a further key process involved in the regulation of this surface receptor.

Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells

Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-beta (TGFbeta) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BRE-luciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.

Differential transcription of Barhl1 homeobox gene in restricted functional domains of the central nervous system suggests a role in brain patterning

The mouse Barhl1 homeogene, member of the BarH subfamily, play a crucial role in the cerebellum development and its human ortholog BARHL1 has been proposed as a positional and functional candidate gene for the Joubert syndrome, a form of cerebellar ataxia. The Barhl1 expression has been demonstrated to be induced by the transcription factor Math1 involved in BMP responses. We isolated the mouse Barhl1 by screening of a cDNA library with the Xenopus Xvent-2, member of the BarH subfamily, which acts in the BMP4 pathway during embryonic patterning and neural plate differentiation. We studied the detailed Barhl1 expression pattern and showed its transcription in spatio-temporally and functionally restricted domains of the developing central nervous system (CNS). Using our new optical microscopy technology, we compare the transcript steady state level and cell density in the Barhl1-expressing regions. We found that Barhl1 was transcribed in superior and inferior colliculi in the dorsal mesencephalon at a relatively low transcriptional level. In the diencephalon, Barhl1 was found higher expressed first within the basal plate and later in the mammillary region. In the cerebellum, Barhl1 showed the highest transcriptional level restricted to the anterior and posterior rhombic lips giving rise to the external and internal cerebellar granular cells and to the deep nuclei. In the spinal cord, Barhl1 showed similar expression level than in the cerebellum and is delimited to a subset of dorsal interneurons. Therefore, our results indicated that Barhl1 homeodomain gene is exclusively transcribed in restricted CNS domain at differential transcription levels which suggest a highly regulated transcriptional mechanism. In addition, these regional and cellular specificities indicated that Barhl1 may be involved in the differentiation of the specific subsets of neuronal progenitors.

SOST/sclerostin, an osteocyte-derived negative regulator of bone formation

Sclerosteosis and Van Buchem disease are two closely related bone disorders characterized by progressive bone thickening due to increased bone formation. Sclerosteosis is associated with mutations in the SOST gene and Van Buchem disease with a 52 kb deletion downstream of the SOST gene that probably affects transcription of the gene. Expression of the gene product sclerostin in bone is restricted to osteocytes and it is a negative regulator of bone formation. It inhibits BMP-stimulated bone formation, but cannot antagonize all BMP responses. The exclusive bone phenotype of good quality of patients with sclerosteosis and Van Buchem disease and the specific localization of sclerostin make it an attractive target for the development of bone forming therapeutics.

Structural Mechanism of Smad4 Recognition by the Nuclear Oncoprotein Ski: Insights on Ski-Mediated Repression of TGF-β Signaling

The Ski family of nuclear oncoproteins represses TGF-β signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-β, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.