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Patients with cystic fibrosis (CF) are at high risk of fat-soluble vitamin deficiencies, even with supplementation. The contribution of a suboptimal vitamin K status to respiratory and endocrine pathophysiology in CF has been inadequately characterized. This is a cross-sectional study in adult CF patients (≥18 years old) from the Montreal Cystic Fibrosis Cohort. Vitamin K1 (VK1) was measured with high-performance liquid chromatography, using fasted serum samples collected during an oral glucose tolerance test (OGTT: 2 h with plasma glucose and insulin every 30 min) ( n = 168). Patients were categorized according to VK1 status (suboptimal defined as INS/GLU ( p = 0.002 and p = 0.006), AUCINS ( p = 0.012) and AUCINS/GLU ( p = 0.004)). Subclinical vitamin K deficiency is more common than other fat-soluble vitamin deficiencies in patients with CF. We demonstrate an association between a suboptimal VK1 status and measures of insulin secretion. We highlight the potential associations of mild vitamin K deficiency with pseudomonal colonization and lower BMI, although these need to be validated in prospective studies.

Pou2af1 encodes for OCA-B, a coactivator of OCT-1/2 transcription factors, which plays a key role in B-cell maturation. The function of OCA-B has also been studied in T cells, where T cells from Pou2af1−/− mice have impaired functions, such as cytokine production and T follicular helper (Tfh) differentiation. Arguably, some of these T-cell phenotypes may result from impaired T–B interactions, secondary to the well-documented B-cell defects in Pou2af1−/− mice. Yet, Pou2af1 is actively transcribed in activated T cells, suggesting a T-cell-intrinsic role. To isolate the T-cell-intrinsic impact of Pou2af1, we generated Pou2af1fl/fl mice with specific genetic disruption of Pou2af1 either in all hematopoietic cells or exclusively in T cells. While we confirm that Pou2af1 is expressed in activated T cells, we surprisingly find that T-cell cytokine production is not impaired in Pou2af1-deficient T cells. Moreover, Pou2af1-sufficient and Pou2af1-deficient T cells have comparable transcriptome profiles, arguing against a T-cell-intrinsic role for Pou2af1. In line with these observations, we demonstrate that Tfh maturation is influenced by T-cell-extrinsic deletion of Pou2af1, as observed both in competitive bone marrow chimeras and in Pou2af1fl/fl mice with specific deletion in B cells. Overall, this study provides strong evidence that Pou2af1 does not act as a transcriptional coactivator in T cells, and conclusively demonstrates that loss of OCA-B in B cells indirectly impacts Tfh differentiation, clarifying the role of OCA-B in the immune system.

Vikas Patel,1 Don Kovalsky,2 S Craig Meyer,3 Abhineet Chowdhary,4 Julie LaCombe,4 Harry Lockstadt,5 Fernando Techy,6 Casey Langel,7 Robert Limoni,8 Philip Yuan,9 Andy Kranenburg,10 Daniel Cher,11 Gabriel Tender12 1Department of Orthopedics, University of Colorado, Aurora, CO, USA; 2Orthopaedic Center of Southern Illinois, Mt. Vernon, IL, USA; 3Columbia Orthopaedic Medical Group, Columbia, MO, USA; 4Overlake Medical Center, Bellevue, WA, USA; 5Bluegrass Orthopaedics, Lexington, KY, USA; 6ClinTech Center for Spine Health, Johnstown, CO, USA; 7The B.A.C.K. Center, Melbourne, FL, USA; 8BayCare Clinic Orthopedics & Sports Medicine, Green Bay, WI, USA; 9Memorial Orthopaedic Surgical Group, Long Beach, CA, USA; 10South Oregon Orthopedics, Medford, OR, USA; 11SI-BONE, Inc, Santa Clara, CA, USA; 12Department of Neurosurgery, Louisiana State University, New Orleans, LA, USACorrespondence: Vikas Patel Tel +1 303 724-8936Email Vikas.Patel@CUAnschutz.eduBackground: Strong evidence supports minimally invasive sacroiliac joint (SIJ) fusion using triangular titanium implants (TTI) for chronic SIJ dysfunction.Objective: To report safety and effectiveness of SIJF using a 3D-printed TTI at 24 months.Methods: SIJF with TTI was performed in 51 subjects. Structured follow-up occurred at 3, 6, 12 and 24 months. Both quality of life questionnaires and functional tests were performed at all study visits.Results: 84% of subjects were available for 24-month follow-up. Observed were rapid and persistent improvements in dysfunction due to pain (Oswestry Disability Index [ODI], mean 52.8 at baseline and 28.3 at 24 months, p< 0.0001) and SIJ pain ratings (mean 78.5 at baseline [0– 100 scale] to 21.5 at 24 months). Opioid use for SIJ pain decreased markedly from baseline. Physical function tests impaired by SIJ pain showed persistent improvements compared to baseline. There was no evidence of device breakage, migration or subsidence and few late adverse events occurred attributable to the device.Conclusion: In this prospective study, SIJF using 3D-printed TTI resulted in immediate, marked and persistent improvements in pain and quality of life, with improved physical function, reduced opioid use and a low rate of late device-related adverse events.Level of Evidence: Level II.Keywords: sacroiliac joint pain, sacroiliac joint arthrodesis, chronic low back pain, triangular titanium implants

SUMMARYVitamin K is a micronutrient necessary for the γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Clinical studies have recently implicated vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse β-cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced β-cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identified Endoplasmic Reticulum Gla Protein (ERGP) as a novel γ-carboxylated ER-resident calcium-binding protein expressed in β-cells. Mechanistically, γ-carboxylation of ERGP protects cells against calcium overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated calcium entry. These results reveal a critical role for vitamin K-dependent γ-carboxylation in the regulation of calcium flux in β-cells and in their capacity to adapt to metabolic stress.

Bone may regulate glucose homeostasis via uncarboxylated bioactive osteocalcin (ucOCN). This study explored whether changes in ucOCN and bone remodeling are associated with change in glucose homeostasis after biliopancreatic diversion (BPD). In this secondary exploratory analysis of a 1-year prospective observational study, 16 participants (11 men/5 women; 69% with type 2 diabetes; mean BMI 49.4 kg/m2) were assessed before, 3 days, 3 months and 12 months after BPD. Changes in plasma ucOCN and bone markers (C-terminal telopeptide (CTX), total osteocalcin (OCN)) were correlated with changes in insulin resistance or sensitivity indices (HOMA-IR; adipose tissue insulin resistance index (ADIPO-IR) and insulin sensitivity index (SI) from the hyperinsulinemic-euglycemic clamp), insulin secretion rate (ISR) from the hyperglycemic clamp, and disposition index (DI: SI × ISR) using Spearman correlations before and after adjustment for weight loss. ucOCN was unchanged at 3 days but increased dramatically at 3 months (+257%) and 12 months (+498%). Change in ucOCN correlated significantly with change in CTX at 3 months (r = 0.62, p = 0.015) and 12 months (r = 0.64, p = 0.025) before adjustment for weight loss. It also correlated significantly with change in fasting insulin (r = −0.53, p = 0.035), HOMA-IR (r = −0.54, p = 0.033) and SI (r = 0.52, p = 0.041) at 3 days, and ADIPO-IR (r = −0.69, p = 0.003) and HbA1c (r = −0.69, p = 0.005) at 3 months. Change in OCN did not correlate with any glucose homeostasis indices. Results were similar after adjustment for weight loss. The increase in ucOCN may be associated with the improvement in insulin resistance after BPD, independently of weight loss. These findings need to be confirmed in larger, less heterogeneous populations.

Bone may regulate glucose homeostasis via uncarboxylated bioactive osteocalcin (ucOCN). This study explored whether changes in ucOCN and bone remodeling are associated with change in glucose homeostasis after biliopancreatic diversion (BPD).In this secondary exploratory analysis of a 1-year prospective observational study, 16 participants (11 men/5 women; 69% with type 2 diabetes; mean BMI 49.4 kg/mucOCN was unchanged at 3 days but increased dramatically at 3 months (+257%) and 12 months (+498%). Change in ucOCN correlated significantly with change in CTX at 3 months (r = 0.62, p = 0.015) and 12 months (r = 0.64, p = 0.025) before adjustment for weight loss. It also correlated significantly with change in fasting insulin (r = -0.53, p = 0.035), HOMA-IR (r = -0.54, p = 0.033) and SI (r = 0.52, p = 0.041) at 3 days, and ADIPO-IR (r = -0.69, p = 0.003) and HbAThe increase in ucOCN may be associated with the improvement in insulin resistance after BPD, independently of weight loss. These findings need to be confirmed in larger, less heterogeneous populations.

The current demographic shift toward an aging population has led to a robust increase in the prevalence of age-associated metabolic disorders. Interestingly, recent studies have demonstrated that the etiology of obesity-related insulin resistance developed upon aging differs from that induced by high-calorie diets. Whereas the role of adaptive immunity on the changes in energy metabolism driven by nutritional challenges has gained attention in the past years, its impact upon aging remains mostly unknown. Here, we found that follicular B2 lymphocytes and expression levels of the B cell-specific transcriptional coactivator OcaB increase with age in spleen and in intra-abdominal epididymal white adipose tissue (eWAT), concomitantly with higher circulating levels of immunoglobulin G (IgG) and impaired glucose homeostasis. Reduction of B cell maturation and immunoglobulin production - especially that of IgG2c - by ablation of OcaB prevented age-induced glucose intolerance and insulin resistance, and promoted energy expenditure by stimulating fatty acid utilization in eWAT and brown adipose tissue. Transfer of wild-type bone marrow in OcaB-/- mice replenished eWAT B2 cell population and IgG levels, which diminished glucose tolerance, insulin sensitivity, and energy expenditure, while increasing body weight gain in aged mice. Thus, these findings demonstrate that upon aging, modifications in B cell-driven adaptive immunity contribute to glucose intolerance and fat accretion.

INTRODUCTION AND OBJECTIVE:Therapeutic options for the management of painful high-tone pelvic floor dysfunction (HTPFD) are limited. We sought to determine whether the addition of pelvic floor chem...

Osteocalcin (OCN) is a bone-derived hormone involved in the regulation of glucose metabolism. In serum, OCN exists in carboxylated and uncarboxylated forms (ucOCN), and studies in rodents suggest that ucOCN is the bioactive form of this hormone. Whether this is also the case in humans is unclear, because a reliable assay to measure ucOCN is not available. Here, we established and validated a new immunoassay (ELISA) measuring human ucOCN and used it to determine the level of bioactive OCN in two cohorts of overweight or obese subjects, with or without type 2 diabetes (T2D). The ELISA could specifically detect ucOCN concentrations ranging from 0.037 to 1.8 ng/mL. In a first cohort of overweight or obese postmenopausal women without diabetes ( n = 132), ucOCN correlated negatively with fasting glucose (r = −0.18, P = 0.042) and insulin resistance assessed by the homeostatic model assessment of insulin resistance (r = −0.18, P = 0.038) and positively with insulin sensitivity assessed by a hyperinsulinemic-euglycemic clamp (r = 0.18, P = 0.043) or insulin sensitivity index derived from an oral glucose tolerance test (r = 0.26, P = 0.003). In a second cohort of subjects with severe obesity ( n = 16), ucOCN was found to be lower in subjects with T2D compared with those without T2D (2.76 ± 0.38 versus 4.52 ± 0.06 ng/mL, P = 0.009) and to negatively correlate with fasting glucose (r = −0.50, P = 0.046) and glycated hemoglobin (r = −0.57, P = 0.021). Moreover, the subjects with ucOCN levels below 3 ng/mL had a reduced insulin secretion rate during a hyperglycemic clamp ( P = 0.03). In conclusion, ucOCN measured with this novel and specific assay is inversely associated with insulin resistance and β-cell dysfunction in humans.

Cell-specific gene inactivation experiments delineate the functions of the enzymes required for osteocalcin modification and demonstrate that it is its uncarboxylated form that acts as a hormone., Osteocalcin (OCN) is an osteoblast-derived hormone favoring glucose homeostasis, energy expenditure, male fertility, brain development, and cognition. Before being secreted by osteoblasts in the bone extracellular matrix, OCN is γ-carboxylated by the γ-carboxylase (GGCX) on three glutamic acid residues, a cellular process requiring reduction of vitamin K (VK) by a second enzyme, a reductase called VKORC1. Although circumstantial evidence suggests that γ-carboxylation may inhibit OCN endocrine functions, genetic evidence that it is the case is still lacking. Here we show using cell-specific gene inactivation models that γ-carboxylation of OCN by GGCX inhibits its endocrine function. We further show that VKORC1 is required for OCN γ-carboxylation in osteoblasts, whereas its paralogue, VKORC1L1, is dispensable for this function and cannot compensate for the absence of VKORC1 in osteoblasts. This study genetically and biochemically delineates the functions of the enzymes required for OCN modification and demonstrates that it is the uncarboxylated form of OCN that acts as a hormone.

Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K-dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1-/- mice survived longer (1 week) than Ggcx-/- mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1-/-;Vkorc1l1-/- mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1-/- mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1-/- mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.

Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its γ-carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function. ispartof: Journal of Clinical Investigation vol:127 issue:11 pages:4104-4117 ispartof: location:United States status: published

The skeleton has recently emerged as an endocrine organ implicated in the regulation of glucose and energy metabolism. This function of bone is mediated, at least in part, by osteocalcin, an osteoblast-derived protein acting as a hormone stimulating insulin sensitivity, insulin secretion and energy expenditure. Osteocalcin secretion and bioactivity is in turn regulated by several hormonal cues including insulin, leptin, the sympathetic nervous system and glucocorticoids. Recent findings support the notion that osteocalcin functions and regulations are conserved between mice and humans. Moreover, studies in mice suggest that osteocalcin could represent a viable therapeutic approach for the treatment of obesity and insulin resistance. In this review, we summarize the current knowledge on osteocalcin functions, its various modes of action and the mechanisms implicated in the control of this hormone.

Vol. 208, No. 6, March 16, 2015. [10.1083/jcb.201409111][1]. The authors noticed a mistake in the labeling of the genotyping primers in Fig. S1 A and Table S1. In Fig. S1 A, the primers P1 and P2 are actually flanking the 5′loxP site region. In addition, the P3 primer should appear downstream of

Osteocalcin is a hormone produced in bones by osteoblasts and regulating energy metabolism. While osteocalcin exists in two forms, γ-carboxylated and undercarboxylated only the latter appears to function as a hormone in vivo. It has been proposed recently that osteoclasts, the bone-resorbing cells, are responsible of decarboxylating, i.e. activating osteocalcin. To address the role of osteoclasts in the maintenance of energy metabolism we analyzed mutant mouse strains harboring either an increase or a decrease in osteoclasts number. Osteoprotegerin-deficient mice that are characterized by an increase in the number of osteoclasts demonstrate an increase in serum levels of undercarboxylated osteocalcin and are significantly more glucose tolerant than WT animals. Conversely, osteoclasts ablation in mice results in a decrease in serum undercarboxylated osteocalcin levels and in reduced glucose tolerance. These results support the notion that osteoclasts are controlling glucose metabolism at least in part through the regulation of osteocalcin decarboxylation.

The clustering of neurons sharing similar functional properties and connectivity is a common organizational feature of vertebrate nervous systems. Within motor networks, spinal motor neurons (MNs) segregate into longitudinally arrayed subtypes, establishing a central somatotopic map of peripheral target innervation. MN organization and connectivity relies on Hox transcription factors expressed along the rostrocaudal axis; however, the developmental mechanisms governing the orderly arrangement of MNs are largely unknown. We show that Pbx genes, which encode Hox cofactors, are essential for the segregation and clustering of neurons within motor columns. In the absence of Pbx1 and Pbx3 function, Hox-dependent programs are lost and the remaining MN subtypes are unclustered and disordered. Identification of Pbx gene targets revealed an unexpected and apparently Hox-independent role in defining molecular features of dorsally projecting medial motor column (MMC) neurons. These results indicate Pbx genes act in parallel genetic pathways to orchestrate neuronal subtype differentiation, connectivity, and organization.

Vitamin K is an essential nutrient involved in the regulation of blood clotting and tissue mineralization. Vitamin K oxidoreductase (VKORC1) converts vitamin K epoxide into reduced vitamin K, which acts as the co-factor for the γ-carboxylation of several proteins, including coagulation factors produced by the liver. VKORC1 is also the pharmacological target of warfarin, a widely used anticoagulant. Vertebrates possess a VKORC1 paralog, VKORC1-like 1 (VKORC1L1), but until very recently, the importance of VKORC1L1 for protein γ-carboxylation and hemostasis in vivo was not clear. Here, we first review the current knowledge on the structure, function and expression pattern of VKORC1L1, including recent data establishing that, in the absence of VKORC1, VKORC1L1 can support vitamin K-dependent carboxylation in the liver during the pre- and perinatal periods in vivo. We then provide original data showing that the partial redundancy between VKORC1 and VKORC1L1 also exists in bone around birth. Recent studies indicate that, in vitro and in cell culture models, VKORC1L1 is less sensitive to warfarin than VKORC1. Genetic evidence is presented here, which supports the notion that VKORC1L1 is not the warfarin-resistant vitamin K quinone reductase present in the liver. In summary, although the exact physiological function of VKORC1L1 remains elusive, the latest findings clearly established that this enzyme is a vitamin K oxidoreductase, which can support γ-carboxylation in vivo.

The only well described function of vitamin K (VK) is to serve as a co-factor for the γ-carboxylation of particular proteins to convert specific glutamic acid (Glu) residues to γ-carboxyglutamic acid (Gla) residues. This process involves two enzymes, γ-glutamyl carboxylase (γ-carboxylase or GGCX) and vitamin K epoxide reductase (VKORC1), which together constitute the vitamin K cycle [1]. Physiologically, γ-carboxylation is known to be critical for the function of many coagulation factors (prothrombin, factor IX, factor VII, etc), but is also implicated in the regulation of Matrix Gla Protein (MGP) that inhibits extra-osseous tissue mineralization and of osteocalcin that regulates glucose metabolism. Osteocalcin is an osteoblast-derived γ-carboxylated protein present at high concentration in the bone extracellular matrix (ECM). Because Gla osteocalcin have great affinity for hydroxyapatite, the mineral component of bone ECM, it was originally believed to play a role in bone mineralization. Nevertheless, gain- and loss-of-function mouse models of osteocalcin have shown that this protein is not required for normal bone remodeling or mineralization [2]. Instead, the characterization of Ocn−/− mice revealed that osteocalcin is acting as a bone-derived hormone regulating glucose metabolism [3-4]. Cell culture and in vivo studies have shown that osteocalcin improves glucose handling by promoting insulin secretion by β-cells on one hand and by favouring insulin sensitivity on the other hand [1]. Although both the Gla and the Glu forms of osteocalcin are detected in the serum, the vast majority of the in vitro and in vivo studies conducted so far suggests that the endocrine function of osteocalcin is fulfilled by its Glu form. Interestingly, additional experiments involving the characterization of mice having either increased or decreased osteoclast number suggest that bone resorption is required to decarboxylate and release osteocalcin from the bone matrix [5–6]. Accordingly, many but not all studies in human have shown that serum levels of Glu osteocalcin negatively correlate with insulin resistance, obesity, diabetes or markers of the metabolic syndrome [1]. So far the data on osteocalcin endocrine regulation were supporting a model in which osteocalcin produced by osteoblasts is stored as a γ-carboxylated and inactive protein in the bone ECM, before being activated by decarboxylation during bone resorption. This model predicts that blocking osteocalcin γ-carboxylation would prevent osteocalcin accumulation in the bone ECM and thereby improve glucose homeostasis. In a recent study published in The Journal of Cell Biology [7] we tested directly this assumption aiming at providing direct evidence demonstrating that γ-carboxylation acts negatively on the endocrine functions of osteocalcin. Mice lacking Ggcx or Vkorc1 globally die perinatally from severe hemorrhages, precluding the study of the role of γ-carboxylation and VK reduction on osteocalcin endocrine function in adults. Hence we generated “floxed” alleles of Ggcx and Vkorc1 in mice in order to be able to inactivate each of these two genes in a tissue-specific manner. Interestingly, mice lacking Ggcx or Vkorc1 in osteoblasts were viable and displayed a marked reduction in Gla osteocalcin serum levels while Glu osteocalcin levels were increased. In addition, osteocalcin failed to accumulate in the bone ECM in the Ggcx and Vkorc1 osteoblast-specific knockout mice. Importantly, these mice also show a significant improvement in glucose tolerance and in insulin sensitivity and were protected from high fat diet-induced insulin resistance and obesity [7]. There is only one γ-carboxylase-encoding gene in mammals; however, all vertebrates possess a second VKORC1-like encoding gene called VKORC1L1. The function of this enzyme remains unclear, but it has been suggested to have VK epoxide reductase activity in vitro, compensating for the absence of VKORC1 in particular cell culture conditions. For this reason, we also generated a conditional allele of this gene in the mouse. Our characterization of the global and osteoblast-specific Vkorc1l1 deficient mice suggests that VKORC1L1 is not implicated in osteocalcin carboxylation. In addition, deleting Vkorc1l1 in osteoblast-specific Vkorc1-deficient mice did not further affect the circulating levels of Glu osteocalcin or osteocalcin bone content when compared to Vkorc1-osteoblasts knockout littermates [7]. These results suggest that VKORC1L1 cannot compensate for VKORC1 absence with respect to γ-carboxylation in osteoblasts. However, it cannot be excluded that VKORC1 and VKORC1L1 may have redundant function(s) in osteoblasts independently of γ-carboxylation. Altogether, these studies support the conclusion that, at least in mice, Glu osteocalcin is the active form of this hormone in vivo and that γ-carboxylation negatively regulates both the bioactivity and the bioavailability of osteocalcin. In addition, this work demonstrates a novel role for vitamin K-dependent γ-carboxylation in the control of glucose metabolism and suggests that VKORC1L1 does not act as a VK reductase in vivo. Future studies will be oriented toward understanding the physiological function of VKORC1L1 in bone and in other tissues.

There is growing concern over confounding artifacts associated with β-cell–specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible β-cell–specific transgenic (MIP-CreERT1Lphi) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in α-cell number and serum glucagon were significantly blunted in MIP-CreERT1Lphi mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for β-cell research.

Introduction Bicycling is associated with neurological impairment and impotence in men. Similar deficits have not been confirmed in women. Aim To evaluate the effects of bicycling on genital sensation and sexual function in women. Methods Healthy, premenopausal, competitive women bicyclists and runners (controls) were compared. Main Outcome Measures (1) Genital vibratory thresholds (VTs) were determined using the Medoc Vibratory Sensation Analyzer 3000. (2) Sexual function and sexually related distress were assessed by the Dennerstein Personal Experience Questionnaire (SPEQ) and the Female Sexual Distress Scale (FSDS). Results Forty‐eight bicyclists and 22 controls were enrolled. The median age was 33 years. The bicyclists were older, had higher body mass indices (BMIs), were more diverse in their sexual orientation, and were more likely to have a current partner. Bicyclists rode an average of 28.3 ± 19.7 miles/day (range 4–100), 3.8 ± 1.5 days/week, for an average of 2.1 ± 1.8 hours/ride. The mean number of years riding was 7.9 ± 7.1 years (range 0.5–30). Controls ran an average of 4.65 ± 2.1 miles/day (range 1.5–8) and 5.0 ± 1.2 days/week. On bivariate analysis, bicyclists had significantly higher VTs than runners, indicating worse neurological function at all sites ( P Conclusion There is an association between bicycling and decreased genital sensation in competitive women bicyclists. Negative effects on sexual function and quality of life were not apparent in our young, healthy premenopausal cohort. Guess MK, Connell K, Schrader S, Reutman S, Wang A, LaCombe J, Toennis C, Lowe B, Melman A, and Mikhail MK. Genital sensation and sexual function in women bicyclists and runners: Are your feet safer than your seat? J Sex Med 2006;3:1018–1027.

The design of new antisense oligomers with improved binding affinity for targeted RNA, while still activating RNase H, is a major research area in medicinal chemistry. RNase H recognizes the RNA–DNA duplex and cleaves the complementary mRNA strand, providing the main mechanism by which antisense oligomers elicit their activities. It has been shown that configuration inversion at the C2' position of the DNA sugar moiety (arabinonucleic acid, ANA), combined with the substitution of the 2'OH group by a fluorine atom (2' F-ANA) increases the oligomer's binding affinity for targeted RNA. In the present study, we evaluated the antisense activity of mixed-backbone phosphorothioate oligomers composed of 2'-deoxy-2'-fluoro-β-D-arabinose and 2'-deoxyribose sugars (S-2' F-ANA–DNA chimeras). We determined their abilities to inhibit the protein expression and phosphorylation of Flk-1, a vascular endothelial growth factor receptor (VEGF), and VEGF biological effects on endothelial cell proliferation, migration, and platelet-activating factor synthesis. Treatment of endothelial cells with chimeric oligonucleotides reduced Flk-1 protein expression and phosphorylation more efficiently than with phosphorothioate antisenses (S-DNA). Nonetheless, these two classes of antisenses inhibited VEGF activities equally. Herein, we also demonstrated the capacity of the chimeric oligomers to elicit RNase H activity and their improved binding affinity for complementary RNA as compared with S-DNA.Key words: antisense DNA, 2' F-ANA nucleosides, mixed-backbone antisense, Flk-1, VEGF.

The osteoblast-derived hormone osteocalcin promotes testosterone biosynthesis in the mouse testis by binding to GPRC6A in Leydig cells. Interestingly, Osteocalcin-deficient mice exhibit increased levels of luteinizing hormone (LH), a pituitary hormone that regulates sex steroid synthesis in the testes. These observations raise the question of whether LH regulates osteocalcin's reproductive effects. Additionally, there is growing evidence that osteocalcin levels are a reliable marker of insulin secretion and sensitivity and circulating levels of testosterone in humans, but the endocrine function of osteocalcin is unclear. Using mouse models, we found that osteocalcin and LH act in 2 parallel pathways and that osteocalcin-stimulated testosterone synthesis is positively regulated by bone resorption and insulin signaling in osteoblasts. To determine the importance of osteocalcin in humans, we analyzed a cohort of patients with primary testicular failure and identified 2 individuals harboring the same heterozygous missense variant in one of the transmembrane domains of GPRC6A, which prevented the receptor from localizing to the cell membrane. This study uncovers the existence of a second endocrine axis that is necessary for optimal male fertility in the mouse and suggests that osteocalcin modulates reproductive function in humans.

In order to resorb the mineralized bone extracellular matrix, the osteoclast relies on the generation of a resorption lacuna characterized by the presence of specific proteases and a low pH. Hence, bone resorption by osteoclasts is highly dependent on lysosomes, the organelles specialized in intra- and extracellular material degradation. This is best illustrated by the fact that multiple forms of human osteopetrosis are caused by mutations in genes encoding for lysosomal proteins. Yet, until recently, the molecular mechanisms regulating lysosomal biogenesis and function in osteoclasts were poorly understood. Here we review the latest developments in the study of lysosomal biogenesis and function in osteoclasts with an emphasis on the transcriptional control of these processes.

SCL/TAL1, a tissue-specific transcription factor of the basic helix-loop-helix family, and c-Kit, a tyrosine kinase receptor, control hematopoietic stem cell survival and quiescence. Here we report that SCL levels are limiting for the clonal expansion of Kit⁺ multipotent and erythroid progenitors. In addition, increased SCL expression specifically enhances the sensitivity of these progenitors to steel factor (KIT ligand) without affecting interleukin-3 response, whereas a DNA-binding mutant antagonizes KIT function and induces apoptosis in progenitors. Furthermore, a twofold increase in SCL levels in mice bearing a hypomorphic Kit allele (W41/41) corrects their hematocrits and deficiencies in erythroid progenitor numbers. At the molecular level, we found that SCL and c-Kit signaling control a common gene expression signature, of which 19 genes are associated with apoptosis. Half of those were decreased in purified megakaryocyte/erythroid progenitors (MEPs) from W41/41 mice and rescued by the SCL transgene. We conclude that Scl operates downstream of Kit to support the survival of MEPs. Finally, higher SCL expression upregulates Kit in normal bone marrow cells and increases chimerism after bone marrow transplantation, indicating that Scl is also upstream of Kit. We conclude that Scl and Kit establish a positive feedback loop in multipotent and MEPs.

Introduction Cycling is associated with genital neuropathies and erectile dysfunction in males. Women riders also have decreased genital sensation; however, sparse information exists addressing the effects of modifiable risks on neurological injuries in females. Aim This study assesses the effects of bicycle setup and cyclists' attributes on GS and saddle pressures among female cyclists. Methods Previously, we compared genital sensation in competitive female cyclists (N = 48) to that of female runners (N = 22). The current study is a subanalysis of the 48 cyclists from the original study group. Nonpregnant, premenopausal women who rode at least 10 miles per week, 4 weeks per month were eligible for participation. Main Outcome Measures Genital sensation was measured in microns using biosthesiometry measures of vibratory thresholds (VTs). Perineal and total saddle pressures were determined using a specialized pressure map and recorded in kilopascals (kPA). Results Handlebars positioned lower than the saddle correlated with increased perineum saddle pressures and decreased anterior vaginal and left labial genital sensation ( P P P P P Conclusion Handlebars positioned lower than the saddle were significantly associated with increased perineum saddle pressures and decreased genital sensation in female cyclists. Modifying bicycle setup may help alleviate neuropathies in females. Additional research is warranted to further assess the extent of the associations. Partin SN, Connell KA, Schrader S, LaCombe J, Lowe B, Sweeney A, Reutman S, Wang A, Toennis C, Melman A, Mikhail M, and Guess MK. The bar sinister: Does handlebar level damage the pelvic floor in female cyclists? J Sex Med 2012;9:1367–1373.

Introduction There are numerous genital complaints in women cyclists, including pain, numbness, and edema of pelvic floor structures. Debate ensues about the best saddle design for protection of the pelvic floor. Aim To investigate the relationships between saddle design, seat pressures, and genital nerve function in female, competitive cyclists. Methods We previously compared genital sensation in healthy, premenopausal, competitive women bicyclists and runners. The 48 cyclists from our original study comprise the study group in this subanalysis. Main Outcome Measures Main outcome measures were: (i) genital vibratory thresholds (VTs) determined using the Medoc Vibratory Sensation Analyzer 3000 and (ii) saddle pressures as determined using a specially designed map sensor. Results More than half of the participants (54.8%) used traditional saddles, and the remainder (45.2%) rode with cut-out saddles. On bivariate analysis, use of traditional saddles was associated with lower mean perineal saddle pressures (MPSP) than riding on cut-out saddles. Peak perineal saddle pressures (PPSP) were also lower; however, the difference did not reach statistical significance. Saddle design did not affect mean or peak total saddle pressures (MTSP, PTSP). Saddle width was significantly associated with PPSP, MTSP, and PTSP but not with MPSP. Women riding cut-out saddles had, on average, a 4 and 11 kPa increase in MPSP and PPSP, respectively, compared with women using traditional saddles (P = 0.008 and P = 0.010), after adjustment for other variables. Use of wider saddles was associated with lower PPSP and MTSP after adjustment. Although an inverse correlation was seen between saddle pressures and VTs on bivariate analysis, these differences were not significant after adjusting for age. Conclusion Cut-out and narrower saddles negatively affect saddle pressures in female cyclists. Effects of saddle design on pudendal nerve sensory function were not apparent in this cross-sectional analysis. Longitudinal studies evaluating the long-term effects of saddle pressure on the integrity of the pudendal nerve, pelvic floor, and sexual function are warranted.

In the developing spinal cord, regional and combinatorial activities of Hox transcription factors are critical in controlling motor neuron fates along the rostrocaudal axis, exemplified by the precise pattern of limb innervation by more than fifty Hox-dependent motor pools. The mechanisms by which motor neuron diversity is constrained to limb levels are, however, not well understood. We show that a single Hox gene, Hoxc9, has an essential role in organizing the motor system through global repressive activities. Hoxc9 is required for the generation of thoracic motor columns, and in its absence, neurons acquire the fates of limb-innervating populations. Unexpectedly, multiple Hox genes are derepressed in Hoxc9 mutants, leading to motor pool disorganization and alterations in the connections by thoracic and forelimb-level subtypes. Genome-wide analysis of Hoxc9 binding suggests that this mode of repression is mediated by direct interactions with Hox regulatory elements, independent of chromatin marks typically associated with repressed Hox genes., National Institutes of Health (U.S.) (P01NS055923)

Abstract 2520 Poster Board II-497 The 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.

Although gross hematuria is a relatively uncommon condition in general obstetrics and gynecology practice, microscopic hematuria is a common incidental finding during routine antepartum or gynecologic office visits. The proper evaluation and treatment options are understudied in females. In fact, work-up of females is controversial, and no consensus guidelines exist at this time. Pregnancy increases the number of potential diagnoses. The majority of published literature on hematuria in pregnancy is in the form of case reports, and esoteric diagnoses are disproportionately represented. The purpose of this review is to summarize existing literature regarding the evaluation, differential diagnosis, and treatment of hematuria in women, with special emphasis on pregnancy and the diagnosis and treatment of microscopic hematuria.

Bicycling is associated with neurological impairment and impotence in men. Similar deficits have not been confirmed in women.To evaluate the effects of bicycling on genital sensation and sexual function in women.Healthy, premenopausal, competitive women bicyclists and runners (controls) were compared.(1) Genital vibratory thresholds (VTs) were determined using the Medoc Vibratory Sensation Analyzer 3000. (2) Sexual function and sexually related distress were assessed by the Dennerstein Personal Experience Questionnaire (SPEQ) and the Female Sexual Distress Scale (FSDS).Forty-eight bicyclists and 22 controls were enrolled. The median age was 33 years. The bicyclists were older, had higher body mass indices (BMIs), were more diverse in their sexual orientation, and were more likely to have a current partner. Bicyclists rode an average of 28.3 +/- 19.7 miles/day (range 4-100), 3.8 +/- 1.5 days/week, for an average of 2.1 +/- 1.8 hours/ride. The mean number of years riding was 7.9 +/- 7.1 years (range 0.5-30). Controls ran an average of 4.65 +/- 2.1 miles/day (range 1.5-8) and 5.0 +/- 1.2 days/week. On bivariate analysis, bicyclists had significantly higher VTs than runners, indicating worse neurological function at all sites (P0.05). Multivariate analysis found significant correlations between higher VTs and bicycling at the left and right perineum, posterior vagina, left and right labia. Increasing VTs at the clitoris, anterior vagina, and urethra were associated with age. In bicyclists, there were no correlations between VTs and miles biked per week, duration of riding, or BMI. Composite SPEQ scores indicated normal sexual function in all sexually active subjects. Neither group suffered from sexually related distress.There is an association between bicycling and decreased genital sensation in competitive women bicyclists. Negative effects on sexual function and quality of life were not apparent in our young, healthy premenopausal cohort.

The objective of this study was to review our experience with pessary use for advanced pelvic organ prolapse. Charts of patients treated for Stage III and IV prolapse were reviewed. Comparisons were made between patients who tried or refused pessary use. A successful trial of pessary was defined by continued use; a failed trial was defined by a patient’s discontinued use. Thirty-two patients tried a pessary; 45 refused. Patients who refused a pessary were younger, had lesser degree of prolapse, and more often had urinary incontinence. Most patients (62.5%) continued pessary use and avoided surgery. Unsuccessful trial of pessary resorting to surgery included four patients (33%) with unwillingness to maintain, three patients (25%) with inability to retain and two patients (17%) with vaginal erosion and/or discharge. Our findings suggest that pessary use is an acceptable first-line option for treatment of advanced pelvic organ prolapse.

Objective To report a rare case of adnexal torsion in an infertile patient with bilateral hydrosalpinges who had proximal tubal occlusion performed before IVF-ET 5 years earlier. Design A case report and literature review. Setting Reproductive endocrine division in a community teaching hospital. Patient(s) A 43-year-old infertile woman with acute left-side pelvic pain. Intervention(s) Laparoscopic bilateral salpingectomies. Main outcome measure(s) Diagnosis and etiology of pelvic pain with subsequent therapy to alleviate symptoms. Result(s) The patient experienced resolution of her pain and prophylactic prevention of further torsion status after bilateral salpingectomies. Conclusion(s) Although laparoscopic proximal tubal occlusion is an effective treatment of hydrosalpinges in patients undergoing IVF-ET, it carries the risk of torsion of the hydrosalpinx and the associated morbidities.

The Saccharomyces cerevisiae prohormone-processing enzyme Kex2p is biosynthesized as an inactive precursor extended by its N-terminal proregion. Here we show that deletion of the proregion renders Kex2p inactive both in vivo and in vitro. Absence of the proregion impaired glycosylation and stability and resulted in the retention of the enzyme in the endoplasmic reticulum. These phenotypes were partially complemented by expression of the proregion intrans. Trans complementation was specific to Kex2p proregion because expression of any of the seven mammalian prohormone convertase propeptides had no effect. These data are consistent with a model whereby Kex2p proregion functions as an intramolecular chaperone and indicate that covalent linkage to the protein is not an absolute requirement for proregion function. Furthermore, extensive mutagenesis revealed that, in addition to their function as proteolytic recognition sites, C-terminal basic residues play an active role in proregion-dependent Kex2p activation.

Hematopoietic stem cells (HSC) reside in specific bone marrow niches comprised of perisinusoidal Nestin-GFP+ (Nes-GFP+) and leptin receptor (LepR)+ stromal cells which highly overlap with each other, as well as osteolineage and endothelial cells. These different cellular constituents regulate HSC maintenance and retention in the bone marrow (BM). Recently, our laboratory further identified rare periarteriolar Nes-GFPbright cells which have been identified to be pericytes and functionally crucial for HSC quiescence and maintenance, from the more abundant reticular Nes-GFPdim population (Kunisaki et al, unpublished data). The function of Nes-GFP+ niche cells is tightly regulated by the sympathetic nervous system (SNS) via β2- and β3-adrenergic receptors. In acute myeloid leukemia (AML), BM infiltration by leukemic blasts is known to lead to hematopoietic failure. Although cytopenias are thought to result from the BM occupation by AML, the mechanisms remain unclear. In this study, we investigated the impact of AML on BM niche constituents, and evaluated the influence of SNS signals in AML progression using a syngeneic murine MLL-AF9 transplantation model. We observed a ∼4.4-fold expansion of Nes-GFP+ stromal cells (p Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Abstract 505 Few markers have thus far been identified on native mesenchymal stem cells (MSCs), both in the mouse and human systems. Most markers cited in the literature are indeed based on expression analyses on heterogeneous cultured cell populations, which may not have self-renewal properties if rigorously tested by transplantation assays. Previous studies using Nestin (Nes)-Gfp transgenic mice showed that Nes-GFP+ cells are self-renewing MSCs, a major constituent of the hematopoietic stem cell (HSC) niche in the bone marrow (BM) (Nature 2010; 466:829). However, the cytoplasmic location of Nestin precludes prospective live cell isolation outside of the transgenic mice. Hence, finding a combination of surface markers labeling Nestin+ cells in situ would be valuable to isolate bona fide MSCs and characterize niche cells. Screening analyses toward this end revealed that PDGFRα and CD51 expression among CD45− Ter119− CD31− BM stromal cells comprised a large fraction (∼60%) of Nes-GFP+ cells. Upon gating first on PDGFRα+ and CD51+, double-positive cells were also highly enriched in Nes-GFP+ cells (∼75%), and represented a rare fraction (∼2%) of the stromal population. Endogenous Nestin expression was also enriched in PDGFRα+ CD51+ cells, compared to single-positive or double-negative stromal cells (control subsets). Cell sorting of BM PDGFRα+ CD51+ and control subsets revealed that PDGFRα+ CD51+ significantly enriched (> 10-fold, p 7-fold, p Disclosures: No relevant conflicts of interest to declare.

SCL/TAL1 is a member of the basic helix-loop-helix family of transcription factors that also includes E2A and HEB. SCL is essential at the onset of hematopoiesis and activates transcription through heterodimerization with E2A. Both genes are expressed in long-term and short-term repopulating hematopoietic stem cells (HSCs) as assessed by expression profiling. To determine whether these genes regulate lineage commitment in HSCs and possibly other HSCs functions, we proceeded to serial transplantation of E2A-deficient or SCL-impaired HSCs. All mice were on a C57Bl/6 background and transplanted donor cells were discriminated from host cells by differential expression of CD45 isoforms. Since SCL function is dosage-sensitive, we used bone marrow cells from SCL LacZ/wt mice in which one SCL allele has been invalidated. When transplanted mice were analyzed at 4 months for reconstitution in all hematopoietic organs, SCLLacZ/wt HSCs (CD45.2) showed normal repopulating capacity in competition with wild type cells (CD45.1) as expected. Strikingly, analysis at 8 months revealed that SCLLacZ/wt HSCs were markedly impaired compared to normal HSCs. The contribution of SCLLacZ/wt cells to the Kit+Sca+Lin− (KSL) and all myeloid progenitor (CMP, MEP and GMP) populations was less than 5%. This long term defect was also observed in secondary transplantation assays. Despite this near absence of HSCs and CMPs, common lymphoid progenitors (CLPs) were almost equally composed of SCLLacZ/wt and wild type cells, and the generation of B22O positive cells was expanded when SCL gene dosage was reduced. Together, our results indicate that long-term stem cell activity requires full SCL gene dosage. Furthermore, SCL operates at the branchpoint of the myeloid and lymphoid lineages to favor the myeloid lineages. Since E2A is an obligate SCL partner, we next addressed E2A function in HSCs. As E2A−/− embryos die before birth, we therefore transplanted E14.5 fetal liver cells. E2A deficiency did not affect the number of HSCs, as assessed by limiting dilution analysis. Nonetheless, serial transplantation assays revealed that HSCs from wild type littermates could be serially transplanted beyond the fourth passage while there was an early exhaustion of E2A-deficient HSCs at the third transplantation. Together, our analyses are consistent with the view that E2A is the functional partner of SCL in HSCs and that SCL/E2A does not control the size of the HSC pool but is required for long-term HSC activity. Similarly to SCL impairment, all progenitors were decreased by E2A deficiency. There were, however, two major differences. First, CLPs were severely impaired indicating that E2A activity is required for specification of the lymphoid lineages. Second, CD11b positive cells were expanded by E2A-deficiency and this was gene dosage-dependent, consistent with the view that E2A inhibits the monocyte-granulocyte lineage. Taken together, our results indicate that SCL collaborates with E2A in HSCs to maintain their long-term activities. In contrast, at the myeloid-lymphoid branchpoint, the functions of SCL and E2A are opposite: SCL favors the myeloid lineage at the expense of the lymphoid lineage, while E2A specifies the lymphoid lineage and inhibits granulocyte-macrophage differentiation.

The c-Kit tyrosine kinase receptor and the SCL/Tal1 (Stem Cell Leukemia) transcription factor are crucial to hematopoiesis and are co-expressed by hematopoietic progenitors. In the present study, we directly address the question whether SCL and c-Kit establish a code for the fate of multipotent progenitors in the adult. We therefore proceeded to loss and gain of function experiments, via retroviral mediated gene transfer in primary hematopoietic cells. We show that SCL is functionally required to sustain the survival of c-Kit+ cells in response to Steel Factor (Kit ligand), but not to GM-CSF and IL-3. Interfering with SCL function using a DNA-binding defective SCL mutant (ΔbSCL) caused a 100-fold loss of multipotent progenitors, due to massive apoptotic death in the c-Kit+ fraction, indicating that the DNA binding function of SCL is essential in cell survival. An antisense SCL also caused apoptosis in c-Kit+ cells, which was rescued by co-delivering SCL in the sense orientation. Furthermore, SCL sets threshold for the response of multipotent progenitors to Steel factor without affecting their response to IL-3 or GM-CSF, demonstrating the specificity of SCL for c-Kit-dependent pathway. Moreover, SCL levels in purified hematopoietic progenitors (HSC, CMP, MEP, CLP) were 3.5 to 15-fold decreased in W41 W41 mice that have a hypomorphic c-Kit allele when compared to age-matched wild type controls. Conversely, ectopic SCL expression is sufficient to bypass c-Kit signalling to suppress apoptosis and increase the proliferative potential of multipotent progenitors, as anemia and other hematopoietic defects caused by a hypomorphic c-Kit allele were rescued by introducing an SCL transgene into the W41 W41 background. We conclude that SCL operates within the c-Kit pathway to suppress apoptosis and determine the clonal output of adult multipotent progenitors. To define the pathway through which SCL suppresses apoptosis in hematopoietic progenitors, we performed microarrays analysis on Δb-SCL expressing cells and identified novel survival genes that were confirmed to be direct SCL targets by chromatin immunoprecipitation. Finally, the expression of these target genes was decreased in purified progenitors from W41 W41 mice as assessed by quantitative PCR. Together, this study reveals a new and essential genetic pathway involving SCL downstream of c-Kit signalling for the survival of hematopoietic progenitors.