Sofie Singbrant

Smad4 is critical for self-renewal of hematopoietic stem cells

Members of the transforming growth factor β (TGF-β) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-β superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4−/− primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal.

Erythropoietin couples erythropoiesis, B-lymphopoiesis, and bone homeostasis within the bone marrow microenvironment

Erythropoietin (Epo) has been used in the treatment of anemia resulting from numerous etiologies, including renal disease and cancer. However, its effects are controversial and the expression pattern of the Epo receptor (Epo-R) is debated. Using in vivo lineage tracing, we document that within the hematopoietic and mesenchymal lineage, expression of Epo-R is essentially restricted to erythroid lineage cells. As expected, adult mice treated with a clinically relevant dose of Epo had expanded erythropoiesis because of amplification of committed erythroid precursors. Surprisingly, we also found that Epo induced a rapid 26% loss of the trabecular bone volume and impaired B-lymphopoiesis within the bone marrow microenvironment. Despite the loss of trabecular bone, hematopoietic stem cell populations were unaffected. Inhibition of the osteoclast activity with bisphosphonate therapy blocked the Epo-induced bone loss. Intriguingly, bisphosphonate treatment also reduced the magnitude of the erythroid response to Epo. These data demonstrate a previously unrecognized in vivo regulatory network coordinating erythropoiesis, B-lymphopoiesis, and skeletal homeostasis. Importantly, these findings may be relevant to the clinical application of Epo.

Canonical BMP signaling is dispensable for hematopoietic stem cell function in both adult and fetal liver hematopoiesis, but essential to preserve colon architecture.

Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5, we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly, concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However, Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover, although BMP receptor expression is increased in fetal liver, fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion, canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis, despite its crucial role in the initial patterning of hematopoiesis in early embryonic development.

Defining the Minimal Factors Required for Erythropoiesis through Direct Lineage Conversion

Summary Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

Endoglin Is Not Critical for Hematopoietic Stem Cell Engraftment and Reconstitution but Regulates Adult Erythroid Development

Endoglin is a transforming growth factor‐β (TGF‐β) accessory receptor recently identified as being highly expressed on long‐term repopulating hematopoietic stem cells (HSC). However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglins role in HSC biology: one that efficiently knocks down expression via lentiviral‐driven short hairpin RNA and another that uses retroviral‐mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin‐suppressed myeloid progenitors (colony‐forming unit‐granulocyte macrophage) displayed a higher degree of sensitivity to TGF‐β‐mediated growth inhibition, whereas endoglin‐overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short‐term or long‐term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast‐forming unit‐erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.

Oncostatin M acting via OSMR, augments the actions of IL-1 and TNF in synovial fibroblasts

OBJECTIVE To identify how the gp130-signaling cytokine oncostatin M (OSM), acting alone or in concert with IL-1β or TNFα, affects synovial fibroblast expression of genes relevant to inflammation and bone erosion in inflammatory arthritis. METHODS Synovial fibroblasts (SFs) were isolated from non-arthritic wild type (WT) or OSM receptor deficient (OSMR(-/-)) mice and stimulated with OSM, IL-1β or TNFα and their combinations. Cytokine gene expression was assessed by quantitative RT-PCR. ELISA, flow cytometry and immunohistochemistry identified protein expression. Gene expression patterns were confirmed in SFs isolated from patients with osteoarthritis (OASFs) and rheumatoid arthritis (RASFs). RESULTS Expression of OSM and its receptors, gp130, OSMR and LIFR, was increased in synovial tissue from the mouse antigen-induced arthritis model. In isolated WT mouse synovial fibroblasts OSM alone, or in synergy with IL-1β, or together with TNFα, potently induced expression of the pro-inflammatory cytokine IL-6. OSM also induced a sustained increase in mRNA levels of the pro-osteoclastic cytokine RANKL. Combining OSM with IL-1β, but not with TNFα, further increased RANKL expression. Importantly these effects of OSM were all dependent on the expression of OSMR. Furthermore, OSM also increased expression of its own receptors, gp130 and OSMR and the IL-1 receptor, IL1-R1; the latter effects were also observed in both human OASFs and RASFs. CONCLUSION Together our data suggests that OSM signaling via OSMR in SFs has the potential to contribute significantly to joint destruction in inflammatory arthritis. It not only induces expression of pro-inflammatory and pro-osteoclastic cytokines but can also augment its own actions and that of IL-1 by inducing expression of OSMR and IL-1R1.

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.

Defining the hematopoietic stem cell niche: the chicken and the egg conundrum

Understanding the in vivo regulation of hematopoietic stem cells (HSCs) will be critical to identifying key factors involved in the regulation of HSC self‐renewal and differentiation. The niche (microenvironment) in which HSCs reside has recently regained attention accompanied by a dramatic increase in the understanding of the cellular constituents of the bone marrow HSC niche. The use of sophisticated genetic models allowing modulation of specific lineages has demonstrated roles for mesenchymal‐derived elements such as osteoblasts and adipocytes, vasculature, nerves, and a range of hematopoietic progeny of the HSC as being participants in the regulation of the bone marrow microenvironment. Whilst providing significant insight into the cellular composition of the niche, is it possible to manipulate any given cell lineage in vivo without impacting, knowingly or unknowingly, on those that remain? J. Cell. Biochem. 112: 1486–1490, 2011.

Gene expression profiling to define the cell intrinsic role of the SKI proto-oncogene in hematopoiesis and myeloid neoplasms.

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced gene signatures associated with hematopoietic stem cells and myeloid differentiation. Here we provide detailed experimental methods and analysis for the gene expression profiling described in our recently published study of Singbrant et al. (2014) in Haematologica. Our data sets (available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39457) provide a resource for exploring the underlying molecular mechanisms of the involvement of the proto-oncogene SKI in hematopoietic stem cell function and development of myeloid neoplasms.

30 ONCOSTATIN M POTENTLY INDUCES IL-6 AND RANKL EXPRESSION IN MOUSE SYNOVIAL FIBROBLASTS AND SIGNIFICANTLY ENHANCES THE EFFECTS OF IL-1 AND TNF

nicely correlated with thickening of the synovial lining layer comprising activated macrophages. When collagenase-induced-osteoarthritis was elicited in S100A9 mice, significantly lower synovial activation was observed when compared to WT mice. Synovial activation was 62% lower at day 42. Cartilage destruction was significantly lower in all surfaces and ranged from a 45% reduction in the lateral tibia to 73% reduction in the medial femur. When primary mouse chondrocytes were stimulated with S100A8 or S100A9, a strong upregulation of particularly MMP-3 mRNA level was found indicating a direct role of S100A8/A9 in cartilage destruction. Conclusions: Alarmins S100A8/S100A9 are expressed by phagocytes in biopsies of early OA patients. S100A8/A9 play a crucial role in synovial activation and cartilage destruction in an osteoarthritis model that shows clear synovial involvement. S100A8/A9 expression in the synovium causes pathology probably by stimulating MMP-mediated damage in the cartilage matrix.