Physiological and regenerative functions of sterile-α motif protein-14 in hematopoiesis.

Sterile α-motif domain-14 (Samd14) protein expression increases the regenerative capacity of the erythroid system. Samd14 is transcriptionally upregulated and promotes cell signaling via the receptor tyrosine kinase Kit in a critical window of acute erythroid regeneration. We generated a hematopoietic-specific conditional Samd14 knockout mouse model (Samd14-CKO) to study the role of Samd14 in hematopoiesis. The Samd14-CKO mouse was viable and exhibited no steady-state hematopoietic phenotype. Samd14-CKO mice were hypersensitive to 5-fluorouracil, resulting in more severe anemia during recovery and impaired erythroid progenitor colony formation. Ex vivo, Samd14-CKO hematopoietic progenitors were defective in their ability to form mast cells. Samd14-CKO mast cells exhibited altered Kit/stem cell factor (SCF), IL-3/IL-3R signaling, and less granularity than Samd14-FL/FL cells. Our findings indicate that Samd14 promotes both erythroid and mast cell functions. The Samd14-CKO mouse phenotype exhibits striking similarities to the KitW/W-v mice, which carry Kit mutations resulting in reduced tyrosine kinase-dependent signaling, causing mast cell and erythroid abnormalities. The Samd14-CKO mouse model is a new tool for studying hematologic pathologies involving Kit signaling.

Ability of metformin to deplete NAD+ contributes to cancer cell susceptibility to metformin cytotoxicity and is dependent on NAMPT expression.

Background: Nicotinamide adenine dinucleotide (NAD+) is vital for not only energy metabolism but also signaling pathways. A major source of NAD+ depletion is the activation of poly (ADP-ribose) polymerase (PARP) in response to DNA damage. We have previously demonstrated that metformin can cause both caspase-dependent cell death and PARP-dependent cell death in the MCF7 breast cancer cells but not in the MDA-MB-231 (231) breast cancer cells while in high-glucose media. We hypothesize that depletion of NAD+ in MCF7 cells via activation of PARP contributes to the cell death caused by metformin. Nicotinamide phosphoribosyltransferase (NAMPT), a key rate-limiting step in converting nicotinamide (vitamin B3) into NAD+, is essential for regenerating NAD+ for normal cellular processes. Evidence shows that overexpression of NAMPT is associated with tumorigenesis. We hypothesize that NAMPT expression may determine the extent to which cancer cells are sensitive to metformin. Results: In this study, we found that metformin significantly decreases NAD+ levels over time, and that this could be delayed by PARP inhibitors. Pretreatment with NAD+ in MCF7 cells also prevents cell death and the enlargement of mitochondria and protects mitochondria from losing membrane potential caused by metformin. This leads to MCF7 cell resistance to metformin cytotoxicity in a manner similar to 231 cells. By studying the differences in NAD+ regulation in these two breast cancer cell lines, we demonstrate that NAMPT is expressed at higher levels in 231 cells than in MCF7 cells. When NAMPT is genetically repressed in 231 cells, they become much more sensitive to metformin-induced cell death. Conversely, overexpressing NAMPT in HEK-293 (293) cells causes the cells to be more resistant to metformin's growth inhibitory effects. The addition of a NAMPT activator also decreased the sensitivity of MCF7 cells to metformin, while the NAMPT activator, P7C3, protects against metformin-induced cytotoxicity. Conclusions: Depletion of cellular NAD+ is a key aspect of sensitivity of cancer cells to the cytotoxic effects of metformin. NAMPT plays a key role in maintaining sufficient levels of NAD+, and cells that express elevated levels of NAMPT are resistant to killing by metformin.

Defining a cohort of anemia-activated cis elements reveals a mechanism promoting erythroid precursor function.

Acute anemia elicits broad transcriptional changes in erythroid progenitors and precursors. We previously discovered a cis-regulatory transcriptional enhancer at the sterile alpha motif domain-14 enhancer locus (S14E), defined by a CANNTG-spacer-AGATAA composite motif and occupied by GATA1 and TAL1 transcription factors, is required for survival in severe anemia. However, S14E is only 1 of dozens of anemia-activated genes containing similar motifs. In a mouse model of acute anemia, we identified populations of expanding erythroid precursors, which increased expression of genes that contain S14E-like cis elements. We reveal that several S14E-like cis elements provide important transcriptional control of newly identified anemia-inducing genes, including the Ssx-2 interacting protein (Ssx2ip). Ssx2ip expression was determined to play an important role in erythroid progenitor/precursor cell activities, cell cycle regulation, and cell proliferation. Over a weeklong course of acute anemia recovery, we observed that erythroid gene activation mediated by S14E-like cis elements occurs during a phase coincident with low hematocrit and high progenitor activities, with distinct transcriptional programs activated at earlier and later time points. Our results define a genome-wide mechanism in which S14E-like enhancers control transcriptional responses during erythroid regeneration. These findings provide a framework to understand anemia-specific transcriptional mechanisms, ineffective erythropoiesis, anemia recovery, and phenotypic variability within human populations.

Functional requirements for a Samd14-capping protein complex in stress erythropoiesis.

Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals-involving cooperation between stem cell factor (SCF)/Kit signaling and other signaling inputs-are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the sterile alpha motif (SAM) domain 14 (Samd14) gene increases the regenerative capacity of the erythroid system in a mouse genetic model and promotes stress-dependent Kit signaling. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. We identified a protein-protein interaction between Samd14 and the α- and ß-heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP ß subunit increased erythroid maturation in murine ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/Kit signaling in CD71med spleen erythroid precursors. Given the roles of Kit signaling in hematopoiesis and Samd14 in Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia.

Anemia is a condition in which the body has a shortage of healthy red blood cells to carry enough oxygen to support its organs. A range of factors are known to cause anemia, including traumatic blood loss, toxins or nutritional deficiency. An estimated one-third of all women of reproductive age are anemic, which can cause tiredness, weakness and shortness of breath. Severe anemia drives the release of hormones and growth factors, leading to a rapid regeneration of precursor red blood cells to replenish the supply in the blood. To understand how red blood cell regeneration is controlled, Ray et al. studied proteins involved in regenerating blood using mice in which anemia had been induced with chemicals. Previous research had shown that the protein Samd14 is produced at higher quantities in individuals with anemia, and is involved with the recovery of lost red blood cells. However, it is not known how the Samd14 protein plays a role in regenerating blood cells, or whether Samd14 interacts with other proteins required for red blood cell production. To shed light on these questions, mouse cells exposed to anemia conditions were used to see what proteins Samd14 binds to. Purifying Samd14 revealed that it interacts with the actin capping protein. This interaction relies on a specific region of Samd14 that is similar to regions in other proteins that bind capping proteins. Ray et al. found that the interaction between Samd14 and the actin capping protein increased the signals needed for the development and survival of new red blood cells. These results identify a signaling mechanism that, if disrupted, could cause anemia to develop. They lead to a better understanding of how our bodies recover from anemia, and potential avenues to treat this condition.