Reduced Expression Of Bmp4 Research Articles

Homeostatic Rho GTPase activation governs thymopoiesis

Abstract The thymus is extremely sensitive to damage, resulting in attenuated T cell reconstitution and failure to elicit appropriate responses to infection, cancer and immunization. Although the thymus has a remarkable regenerative capacity, the mechanisms underlying this endogenous regeneration remain poorly understood. Previously we have identified two critical secreted factors that drive thymic regeneration, including BMP4 from endothelial cells (ECs) and IL-22 from dendritic cells (ILCs), which can be used as therapeutic regenerative strategies. Additionally, we have shown that steady-state CD4+CD8+ thymocytes (DPs) suppress levels of IL-23 (Science 336:91), a key regulator of IL-22; and as developing thymocytes undergo high levels of apoptosis, we hypothesize that apoptotic DPs mediate suppression of regenerative factors under homeostatic conditions, and damage-induced DP depletion removes this suppression, triggering thymic recovery. Here, we demonstrate a DP-specific reduction in cl-caspase 3 levels after acute injury in mice (total body irradiation, 550 cGy), and reduced Bmp4 expression in ECs co-cultured with apoptotic thymocytes, an effect rescued by apoptosis inhibition, using the pan-caspase inhibitor zVAD-FMK. We identified that apoptotic thymocytes mediate their suppression via TAM receptors in ECs and DCs, which subsequently activate Rho GTPases. Importantly, we show that inhibition of Rac1 activation increases Bmp4 and Il23 expression in ECs and DCs, respectively, and in vivo inhibition of Rac1 with EHT1864 enhances thymus cellularity in models of acute injury and age. These data describe a novel regulatory pathway that can be therapeutically targeted to boost T cell reconstitution after damage.

Homeostatic Regulation of Apoptosis Governs Thymus Regeneration

Although the thymus has a remarkable capacity for repair following acute injury, such as that caused by the conditioning required for successful hematopoietic cell transplant (HCT), the mechanisms underlying this endogenous regeneration remain poorly understood. Delayed T cell reconstitution occurs following thymus insult and can exceed more than a year post-transplant due to a delay in full recovery of thymic output, function and T cell repertoire. Therefore, strategies to enhance T cell reconstitution post-transplant represents a rational approach to significantly improve the overall outcome of allo-HCT. We propose that enhancing thymic function will boost T cell reconstitution and substantially increase immune responses following allo-HCT. Our recent studies have identified two critical pathways that govern thymic regeneration; centered on secretion of BMP4 by endothelial cells (ECs) and IL-22 by innate lymphoid cells (Dudakov 2012 Science 336:91; Dudakov 2017 Blood 130:933; Wertheimer 2018 Sci Immunol 3:19). However, the specific regulatory mechanisms that trigger these regeneration-associated factors (RAFs) after damage remain unclear. Given that our prior work revealed that the presence of DP thymocytes suppresses the production of RAFs like IL-23, a key downstream mediator of IL-22; and the high basal rate of thymocyte apoptosis, as apoptotic thymocytes form the bulk of developing T cells, we hypothesized that apoptotic DP thymocytes were mediating this suppression of RAFs under homeostatic conditions. Upon injury, loss of DP thymocytes leads to reduced apoptotic signaling and reduced suppression of RAFs, triggering thymic recovery (Fig 1A). Consistent with this hypothesis, our preliminary data shows a significantly reduced number of apoptotic thymocytes after total body irradiation (TBI, 550 cGy), as measured by cleaved caspase 3 levels (Fig 1B). Additionally, co-culture of apoptotic thymocytes results in reduced Bmp4 expression in ECs, which is rescued by inhibition of thymocyte apoptosis using the pan-caspase inhibitor zVAD-FMK (Fig 1C). One way in which apoptotic thymocytes could induce this suppression of RAFs is via TAM receptor activation, which is supported by our data demonstrating increased Bmp4 expression in ECs treated with a pan-TAM receptor antagonist and subsequently co-cultured with apoptotic thymocytes (Fig 1D). Interestingly, TAM receptors can activate Rac1, a Rho GTPases involved in actin cytoskeletal rearrangement; converging neatly on our previous data showing that inhibition of Rac1 with small molecule inhibitors led to robust induction of Bmp4 and Il23 expression. Therefore, we propose that in steady-state, apoptotic thymocytes activate TAM receptors on ECs and DCs and induce intracellular activation of Rac1, which ultimately suppresses the production of BMP4 and IL-23; but after damage, when the number of apoptotic thymocytes drops precipitously, this suppression is abrogated, allowing for thymic regeneration (Fig 1E). Importantly, we demonstrate here that this pathway can be therapeutically targeted, as inhibition of Rac1 in vivo with EHT1864 enhances thymus cellularity in models of acute injury (Fig. 1F), and age (Fig. 1G). As post-transplant T cell deficiency is associated with an increased risk of infections, relapse of malignancy, and the development of secondary malignancies, identifying molecular targets to enhance thymic recovery will aid in the development of therapeutics with imminent clinical need. These findings not only reveal a novel molecular mechanism governing tissue regeneration, but also offer a potentially superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by allo-HCT, infection or cytoreductive therapy. Disclosures No relevant conflicts of interest to declare.

Loss of peri-Wolffian duct stromal Frs2α expression in mice leads to abnormal ureteric bud induction and vesicoureteral reflux.

BackgroundFibroblast growth factor receptor 2 (Fgfr2) deletion from murine peri-Wolffian duct stroma (ST) results in aberrant ureteric bud induction, abnormal ureteral insertion into the bladder, and high rates of vesicoureteral reflux (VUR). It is unclear which receptor docking protein(s) is/are responsible for Fgfr2 actions in these tissues. We investigated whether the docking protein, fibroblast receptor substrate 2α (Frs2α), had roles in peri-Wolffian duct stroma similar to Fgfr2.MethodsWe conditionally deleted Frs2α in per-Wolffian duct stroma with a Tbx18cre mouse line (Frs2αST−/−). We assessed for ureteric induction defects and alterations in downstream targets mediating defects. We performed euthanized cystograms and assessed ureter-bladder junctions via three dimensional (3D) reconstructions.ResultsEmbryonic day (E) 11.5 Frs2αST−/− embryos had many displaced ureteric bud induction sites versus controls. E11.0 Frs2αST−/− embryos had decreased Bmp4 expression and signaling, which can cause abnormal ureteric bud induction. Postnatal day 1 (P1) and P30 Frs2αST−/− mice had higher VUR rates and grades versus controls. Mutant refluxing ureters inserted improperly into the bladder and had shortened intravesicular tunnels versus controls:ConclusionsFrs2αST−/− have aberrant ureteric induction sites, improper ureteral insertion, shortened intravesicular lengths and VUR. Induction site defects appear secondary to reduced Bmp4 expression, similar to Fgfr2 mutants.

Epigenomic Silencing of the BMP-4 Gene in Pituitary Adenomas: A Potential Target for Epidrug-Induced Re-expression

Bone morphogenetic protein (BMP)-4 is a key mediator of anterior pituitary organogenesis. However, through inappropriate expression patterns, BMP-4 is also pathogenic in a pituitary adenoma subtype-specific context. In these cases, increase or decrease in BMP-4 in lactotroph- and corticotroph-derived adenomas, respectively, is consistent with a bifunction role for this protein toward either promotion or inhibition of cell proliferation and hormone secretion. To gain insight into the aberrations responsible for differential expression, we examined BMP-4 transcript and protein expression patterns in the major adenomas subtypes. BMP-4 transcript and protein are differentially expressed and show increase in the majority of prolactinomas relative to normal pituitary, whereas the majority of other adenoma subtypes show reduced expression relative to both prolactinoma and normal pituitaries. Reduced expression of BMP-4 is not associated with change in CpG island methylation status. However, histone tail modifications are apparent, as enrichment for a modification associated with silent genes, H3K27me3, and depletion of a modification associated with active genes, H3K9Ac. In pituitary cell lines, reduced BMP-4 expression is also associated with similar histone tail modifications and contemporaneous increase in CpG island methylation. In these cells, coincubation with the demethylating agent zebularine and histone deacetylase inhibitor, trichostatin A, reversed epigenetic changes and restored expression of BMP-4. These studies show that, in contrast to prolactinomas, other adenoma subtypes show reduced expression of BMP-4 where epidrug induced reexpression, alone or in combination with conventional therapies, may offer new treatment strategies.

Reduced BMP4 abundance in Gata2 hypomorphic mutant mice result in uropathies resembling human CAKUT

Constitutive loss of transcription factor GATA-2 leads to embryonic lethality from primitive erythropoietic failure. We serendipitously discovered an essential contribution of GATA-2 to urogenital development when the hematopoietic deficiency of Gata2 null mutant animals was complemented by a Gata2 yeast artificial chromosome (YAC) transgene; these mice died from a perinatal lethal urogenital abnormality. Here, we report the generation and analysis of Gata2 hypomorphic mutant (Gata2(fGN)/(/fGN)) mice, which suffered from hydronephrosis and megaureter, as do the YAC-rescued Gata2 null mutants. Gata2(fGN)/(/fGN) mutants exhibit anteriorly displaced ureteric budding from the Wolffian duct as well as reduced BMP4 expression in the intermediate mesoderm derivatives in a manner that is temporally coincident with ureteric bud emergence. In Bmp4 mutant heterozygotes, rostral displacement of the initial bud site on the Wolffian duct results in abnormal urogenital development. We show here that Bmp4 mRNA is reduced approximately twofold in Gata2(fGN)/(/fGN) mice (as in Bmp4 null heterozygotes), and that GATA-2 trans-activates a Bmp4 first intron element-directed reporter plasmid in co-transfection assays. These experiments taken together implicate GATA-2 as a direct regulator of Bmp4 transcription. The pathophysiology described in Gata2 hypomorphic mutant animals resembles human congenital anomalies of the kidney and urinary tract.

Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation

Msx and Dlx homeoproteins control the morphogenesis and organization of craniofacial skeletal structures, specifically those derived from the pharyngeal arches. In vitro Msx and Dlx proteins have opposing transcriptional properties and form heterodimeric complexes via their homeodomain with reciprocal functional repression. In this report we examine the skeletal phenotype of Msx1; Dlx5 double knock-out (DKO) mice in relationship with their expression territories during craniofacial development. Co-expression of Dlx5 and Msx1 is only observed in embryonic tissues in which these genes have independent functions, and thus direct protein interactions are unlikely to control morphogenesis of the cranium. The DKO craniofacial phenotypes indicate a complex interplay between these genes, acting independently (mandible and middle ear), synergistically (deposition of bone tissue) or converging on the same morphogenetic process (palate growth and closure). In the latter case, the absence of Dlx5 rescues in part the Msx1-dependent defects in palate growth and elevation. At the basis of this effect, our data implicate the Bmp (Bmp7, Bmp4)/Bmp antagonist (Follistatin) signal: in the Dlx5−/− palate changes in the expression level of Bmp7 and Follistatin counteract the reduced Bmp4 expression. These results highlight the importance of precise spatial and temporal regulation of the Bmp/Bmp antagonist system during palate closure.

Genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal repressor.

According to a model based on embryological studies in amphibia, dorsoventral patterning is regulated by the antagonizing function of ventralizing bone morphogenetic proteins (BMPs) and dorsalizing signals generated by Spemann's organizer. Large-scale mutant screens in the zebrafish, Danio rerio, have led to the isolation of two classes of recessive lethal mutations affecting early dorsoventral pattern formation. dino mutant embryos are ventralized, whereas swirl mutants are dorsalized. We show that at early gastrula stages, dino and swirl mutants display an expanded or reduced Bmp4 expression, respectively. The dino and swirl mutant phenotypes both can be phenocopied and rescued by the modulation of BMP signaling in wild-type and mutant embryos. By suppressing BMP signaling in dino mutants, adult fertile dino -/- fish were generated. These findings, together with results from the analysis of dino-swirl double mutants, indicate that dino fulfills its dorsalizing activity via a suppression of swirl-dependent, BMP-like ventralizing activities. Finally, cell transplantation experiments show that dino is required on the dorsal side of early gastrula embryos and acts in a non-cell-autonomous fashion. Together, these results provide genetic evidence in support of a mechanism of early dorsoventral patterning that is conserved among vertebrate and invertebrate embryos.