Role Of SCL Research Articles

1964-P: Type 1 Diabetes: Sclerostin Resistance Improves Glucose Metabolism and Protects Bone Mass

Type 1 diabetes (T1D) is characterized by metabolic abnormalities and bone fragility. In humans and preclinical models of T1D, serum markers of bone formation are decreased suggesting reduced bone turnover rates. Moreover, circulating levels of the Wnt inhibitor sclerostin (Scl) are higher in T1D patients. To test the role of Scl in T1D bone disease, we introduced the Scl-resistant Lrp5A214V mutation, associated with high bone mass (HBM), in mice carrying the Ins2Akita mutation (Akita), which causes hypoinsulinemia and hyperglycemia at age 4-5 weeks. Although both Akita and Akita/HBM mutants developed diabetes (non-fasting blood glucose ≥300 mg/dl), they showed a very different onset timing. At 6 weeks, only 30% of Akita/HBM mice developed hyperglycemia, compared to 90% of Akita mice (n=10), with the majority of Akita/HBM mice becoming hyperglycemic by 12 weeks. At 6 weeks, Akita mice showed a significant impairment in glucose tolerance in an intraperitoneal glucose tolerance test (ipGTT) relative to mild glucose intolerance demonstrated in Akita/HBM mice (p<0.05 for difference in AUC; n=5-7). No differences in serum insulin, glucagon and C-peptide were found between Akita/HBM and Akita mice at time 0 and 30 minutes during the ipGTT (n=2-6). Importantly however, Akita/HBM showed a significant improvement in insulin sensitivity in an ip insulin tolerance test relative to Akita mice at 7 weeks of age, which persisted until 30 weeks (p<0.05 for difference in AUC; n= 4-7). Strikingly, bone mass was 18% higher in Akita/HBM relative to Akita littermates at 12 weeks (p<0.001; n=3-6), a difference that persisted up until at least 18 weeks of age, despite the prolonged hyperglycemia. Together, these results suggest that Scl resistance may improve peripheral glucose metabolism and slow down the onset of T1D, although it does not prevent it. Even though we have not yet measured bone strength, Wnt stimulation protects bone mass and may delay the onset of metabolic abnormalities seen in T1D. Disclosure G. Leanza: None. Y. Alippe: None. S. Lee: None. R. Strollo: None. P. Pozzilli: None. R. Civitelli: Stock/Shareholder; Self; Amgen Inc., Eli Lilly and Company, Merck & Co., Inc. N. Napoli: None.

SCL/TAL1 Regulates Hematopoietic Specification From Human Embryonic Stem Cells

Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45(-)CD31(+)CD34(+)) and, to a lesser extent, on CD45(+) hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34(+)CD45(+)) and total (CD45(+)) blood cells with higher clonogenic potential. Short-hairpin RNA-based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.

SCL regulates the Quiescence and the Long-Term Competence of Hematopoietic Stem Cells.

Abstract 2520Poster Board II-497The life-long production of blood cells depends on the regenerative capacity of a rare bone marrow population, the hematopoietic stem cells (HSCs). In the adult, the majority of HSCs are quiescent while a large proportion of progenitors are more cycling. The state of quiescence in HSCs is reversible and these cells can be triggered into cycle by chemotoxic injuries, exposure to cytokines in vitro, as well as transplantation in vivo. SCL/TAL1 is a bHLH transcription factor that has a critical role in generating HSCs during development. However, the role of SCL in adult HSCs is still a matter of debate. In the present study, we took several approaches to address this question. Scl expression was monitored by quantitative PCR analysis in a population that contains adult long-term reconstituting HSCs (LT-HSCs) at a frequency of 20–50%: Kit+Sca+Lin-CD150+CD48-. RT-PCR results were confirmed by β-galactosidase staining of these cells in Scl-LacZ mice. We show that Scl is highly expressed in LT-HSC and that its expression correlates with quiescence, i.e. Scl levels decrease when LT-HSCs exit the G0 state. In order to assess stem cell function, we performed several transplantation assays with adult bone marrow cells in which SCL protein levels were decreased at least two-fold by gene targeting or by RNA interference. 1) The mean stem cell activity of HSCs transplanted at ∼1 CRU was two-fold decreased in Scl heterozygous (Scl+/−) mice. 2) In competitive transplantation, the contribution of Scl+/− cells to primitive populations as well mature cells in the bone marrow was significantly decreased 8 months after transplantation. 3) In secondary transplantation assays, Scl+/− HSCs were severely impaired in their ability to reconstitute secondary recipient in stem cells and progenitor populations and in almost all mature lineages. 4) Reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was significantly decreased compared to controls. We therefore conclude that SCL levels regulate HSC long term competence. Since Scl levels decrease when LT-HSCs exit the G0 state, we addressed the question whether the cell cycle state of LT-HSCs is sensitive to Scl gene dosage. We stained bone marrow cell populations with Hoechst and Pyronin Y. At steady state, percentage LT-HSCs in G1 fraction appears to be significantly increased in mice lacking one allele of Scl. Furthermore, a three-fold increase in G1 fraction was also observed when cells were infected with Scl-directed shRNA, suggesting that a decrease in Scl levels facilitates G0-G1 transition. At the molecular level, we show by chromatin immunoprecipitation that SCL occupies the Cdkn1a and Id1 loci. Furthermore, in purified Kit+Sca+Lin-CD150+CD48- cells, the expression levels of these two regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence. Disclosures:No relevant conflicts of interest to declare.

SCL Expression at Critical Points in Human Hematopoietic Lineage Commitment

The stem cell leukemia (SCL or tal-1) gene was initially identified as a translocation partner in a leukemia that possessed both lymphoid and myeloid differentiation potential. Mice that lacked SCL expression showed a complete block in hematopoiesis; thus, SCL was associated with hematopoietic stem cell (HSC) function. More recent studies show a role for SCL in murine erythroid differentiation. However, the expression pattern and the role of SCL during early stages of human hematopoietic differentiation are less clear. In this study we chart the pattern of human SCL expression from HSCs, through developmentally sequential populations of lymphoid and myeloid progenitors to mature cells of the hematopoietic lineages. Using recently defined surface immunophenotypes, we fluorescence-activated cell-sorted (FACS) highly purified populations of primary human hematopoietic progenitors for reverse transcription-polymerase chain reaction (RT-PCR) analysis of SCL expression. Our data show that SCL mRNA is easily detectable in all hematopoietic populations with erythroid potential, including HSCs, multipotential progenitors, common myeloid progenitors, megakaryocyte/erythrocyte progenitors, and nucleated erythroid lineage cells. SCL mRNA expression was present but rapidly downregulated in the common lymphoid progenitor and granulocyte/monocyte progenitor populations that lack erythroid potential. SCL expression was undetectable in immature cells of nonerythroid lineages, including pro-B cells, early thymic progenitors, and myeloid precursors expressing the M-CSF receptor. SCL expression was also absent from all mature cells of the nonerythroid lineages. Although low levels of SCL were detected in lymphoid- and myeloid-restricted progenitors, our studies show that abundant SCL expression is normally tightly linked with erythroid differentiation potential.

Stem cell leukemia protein directs hematopoietic stem cell fate

AbstractStem cell leukemia (SCL) protein has been shown to be an essential transcription factor during hematopoietic development in the embryo. In adult hematopoiesis, however, the role for SCL has remained largely unknown, whereas it is expressed in bone marrow hematopoietic stem cells (HSCs). In this study, we performed HSC transplantation and an in vitro HSC differentiation assay using retrovirally transduced HSCs with wild-type (WT) and dominant-negative (DN) SCL. The transplantation experiments showed that SCL does not affect the long-term repopulating capacity of HSCs but that WT SCL and DN SCL increase the short-term contribution of the transduced HSCs in myeloid and lymphoid lineages, respectively. An in vitro single-cell assay using a fetal thymus organ culture system further demonstrated that WT SCL facilitates HSCs to differentiate into the myeloid lineage but that DN SCL facilitates HSCs to differentiate into the lymphoid lineage. We conclude that the up-regulation or down-regulation of SCL directs HSCs toward myeloid or lymphoid lineage, respectively, although SCL does not affect their long-term repopulating capacity. (Blood. 2004;103: 3336-3341)

The SCL gene specifies haemangioblast development from early mesoderm.

The SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of all haematopoietic lineages. SCL is also expressed in endothelial cells, but its function is not essential for specification of endothelial progenitors and the role of SCL in endothelial development is obscure. We isolated the zebrafish SCL homologue and show that it was co-expressed in early mesoderm with markers of haematopoietic, endothelial and pronephric progenitors. Ectopic expression of SCL mRNA in zebrafish embryos resulted in overproduction of common haematopoietic and endothelial precursors, perturbation of vasculogenesis and concomitant loss of pronephric duct and somitic tissue. Notochord and neural tube formation were unaffected. These results provide the first evidence that SCL specifies formation of haemangioblasts, the proposed common precursor of blood and endothelial lineages. Our data also underline the striking similarities between the role of SCL in haematopoiesis/vasculogenesis and the function of other bHLH proteins in muscle and neural development.

Steel factor affects SCL expression during normal erythroid differentiation

Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E- derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel.

SCL and related hemopoietic helix-loop-helix transcription factors.

The helix-loop-helix (HLH) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals. Five members of this family (MYC, SCL, TAL-2, LYL-1 and E2A) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations. Although activated in T cell leukemias, expression of SCL and LYL-1 is low or undetectable in normal T cell populations. SCL is expressed in erythroid, megakaryocyte and mast cell populations (the same cell lineages as GATA-1, a zinc-finger transcription factor). In addition, both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells, thus implying a role for SCL in erythroid differentiation events. However, in contrast to GATA-1, SCL is expressed in the developing brain. Studies of the function of SCL suggest it is also important in proliferation and self-renewal events in erythroid cells.