RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family with unknown physiological function. Here we demonstrate that Rhoh-/- mice have impaired T cell receptor (TCR)-mediated thymocyte selection and maturation, resulting in T cell deficiency. RhoH deficiency resulted in defective CD3zeta phosphorylation, impaired translocation of the signaling molecule Zap70 to the immunological synapse and reduced activation of Zap70-mediated signaling in thymic and peripheral T cells. Proteomic analyses demonstrated that RhoH is a component of TCR signaling and is required for recruitment of Zap70 to the TCR through interaction with RhoH noncanonical immunoreceptor tyrosine-based activation motifs (ITAMs). In vivo reconstitution studies also demonstrated that RhoH function depends on phosphorylation of the RhoH ITAMs. These findings suggest that RhoH is a critical regulator of thymocyte development and TCR signaling by mediating recruitment and activation of Zap70.
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) is the key enzyme in melatonin synthesis regulated by circadian rhythm. To date, our understanding of the oscillatory mechanism of melatonin has been limited to autoregulatory transcriptional and posttranslational regulations of AANAT mRNA. In this study, we identify three proteins from pineal glands that associate with cis-acting elements within species-specific AANAT 3 untranslated regions to mediate mRNA degradation. These proteins include heterogeneous nuclear ribonucleoprotein R (hnRNP R), hnRNP Q, and hnRNP L. Their RNA-destabilizing function was determined by RNA interference and overexpression approaches. Expression patterns of these factors in pineal glands display robust circadian rhythm. The enhanced levels detected after midnight correlate with an abrupt decline in AANAT mRNA level. A mathematical model for the AANAT mRNA profile and its experimental evidence with rat pinealocytes indicates that rhythmic AANAT mRNA degradation mediated by hnRNP R, hnRNP Q, and hnRNP L is a key process in the regulation of its circadian oscillation.Circadian rhythm is a fundamental biological phenomenon in living organisms (10,41,53). To date, efforts to understand the molecular mechanisms of circadian rhythm have focused mainly on transcriptional regulation. A number of studies show that autoregulatory transcriptional-posttranslational feedback loops are crucial for the rhythmic expression of clock-controlled genes (14,30,40,41,46). However, limited data on the posttranscriptional level are available (45). Since mRNA turnover has notable effects on the synthesis of specific proteins and provides the cell with flexibility in achieving rapid changes at the transcript level (9, 35, 50, 52), it is possible that posttranscriptional regulation functions in the rhythmic expression of circadian genes.Recent evidence supports the existence of posttranscriptional mechanisms. In Drosophila, the degradation of Period (per) mRNA modulates its proper circadian fluctuation (49). The accelerated decay of mouse Per1 (mPer1) mRNA in a tau mutant is additionally suggestive of the presence of a posttranscriptional regulatory pathway (32). In transgenic experiments, the differences between the mRNA fluctuations of clock-controlled genes and reporters were tentatively accounted for by variations in their mRNA stability mediated by 3Ј untranslated regions (3ЈUTRs) (22, 51). In computational modeling approaches, mRNA degradation is assumed in the construction of circadian clock models, although its role in rhythm formation is not currently clear (12, 31). Here, we postulate that dynamic mRNA degradation is essential for the formation of circadian rhythms in clock-controlled gene expression, and we support our theory with mathematical modeling and experimental evidence of rat serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) mRNA rhythms.AANAT is a rate-limiting enzyme in the melatonin synthetic pathway that drives the daily rhythm in the leve...
SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1 ؊/؊ mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1 ؊/؊ mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1 ؉/؉ , ؉/؊ , and ؊/؊ mice. SIRT1 ؊/؊ ESCs formed fewer mature blast cell colonies. Replated SIRT1 ؊/؊ blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1 ؊/؊ -derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1 ؊/؊ ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased -H1 globin, -major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1 ؊/؊ ESC differentiation deficiencies. SIRT1 ؊/؊ yolk sacs manifested fewer primitive erythroid precursors. SIRT1 ؊/؊ and SIRT1 ؉/؊ adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis. (Blood. 2011;117(2):440-450) IntroductionMouse embryonic stem cells (ESCs) are pluripotent with capacity for unlimited self-renewal or differentiation into endoderm, ectoderm, and mesoderm. Self-renewal behavior in vitro is sustained with leukemia inhibitory factor (LIF). 1 With removal of LIF and in the absence of feeder layer cells, ESCs grow into spheres termed embryoid bodies (EBs), which generate hematopoietic and endothelial progeny recapitulating development of those populations in the yolk sac. 2 Hemangioblasts generate blast colonies in vitro displaying hematopoietic and endothelial potential. 3 The ESC/EB system provides a powerful in vitro model to explore cellular and molecular events that specify lineage choice and hematopoietic commitment.Sirtuins, or Sir2 family proteins, are conserved from bacteria to humans. 4 Sir2 modulates longevity and aging in yeast, Caenorhabditis elegans, and Drosophila. 5 Mammalian homologs of Sir2 encompass a family of 7 proteins (SIRT1-SIRT7), among which SIRT1 is the closest human homolog of the yeast Sir2 protein. 4 SIRT1 deacetylates proteins, including p53 and FOXO transcription factors, and plays many key functions including energy metabolism, differentiation, aging, and tumor suppression. [6][7][8][9][10] SIRT1 is expressed at high levels in mouse embryos with the highest SIR2␣ mRNA expression is embryonic day (E) 4.5 embryos. Although expression is down-regulated during subsequent embryogenesis, high level expression rema...
IntroductionRho guanosine triphosphatases (GTPases), belonging to the Ras superfamily, play key roles in regulating actin organization, membrane trafficking, migration, and cell adhesion. 1 The activity of most Rho GTPases is characterized by reversible binding of guanosine triphosphate (GTP). The cycling of Rho GTPases between GTP-bound, active and guanosine diphosphate-bound, inactive forms is regulated by distinct cellular proteins including guanine nucleotide exchange factors and GTPase activating proteins (GAPs). 2,3 Rac subfamily members, Rac1 and Rac2, are essential for cortical filamentous-actin (F-actin) assembly and chemotaxis in hematopoietic stem/progenitor cells. 4,5 Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which is associated with loss of adhesion, chemotaxis, and cortical F-actin polymerization. 5 Rhoh was first identified as a fusion transcript with LacZ3/Bcl6 in non-Hodgkin lymphoma 6 and encodes a hematopoietic-specific member of the RhoE/Rnd3 subfamily that lacks intrinsic and/or agonist-induced GTPase activity. Therefore, RhoH remains in a GTP-bound, constitutively active state without cycling. The cellular activity of RhoH has been postulated to be regulated by cell-specific transcription that determines its protein level and posttranslational modification. 7-9 Alteration of RhoH expression level disrupts normal proliferation, survival, and engraftment of HPCs in lethally irradiated recipient mice, as well as cortical F-actin assembly and chemokine-induced migration. 10 Overexpression of RhoH also impairs the activation of Rac GTPases in HPCs 10 and inhibits Rac1-mediated downstream signaling pathways in T cells. 7 These findings suggest that RhoH may antagonize Rac function in regulating some cellular processes. However, the molecular mechanisms underlying the crosstalk between Rac and RhoH have not been defined.Rho GTPases associate with effector proteins at the cell plasma membrane, including lipid rafts. 11-15 Detachment of cells induces the internalization of lipid rafts, which inhibits the membrane localization of Rac1. In addition, cell-permeable C-terminal peptides of Rac1 localize to plasma membrane in HL60 cells and inhibit Rac1 membrane localization. This is associated with impaired actin polymerization and cell migration. 12 Thus, translocation of intracellular Rac proteins to the plasma membrane is critical for activating Rac-mediated downstream signaling and function. 16 Rac GTPases contain a conserved C-terminal CAAX motif, which is posttranslationally modified by isoprenylation of the cysteine residue. The isoprenoid moiety is required to anchor Rac proteins to the cell membrane. 17 In this study, we examined the biologic role of RhoH in actin cytoskeleton organization and chemotaxis in response to stromalderived factor-1␣ (SDF-1␣) using freshly isolated Rhoh Ϫ/Ϫ HPCs. Loss of RhoH in these cells causes enhanced Rac-mediated migration and cortical F-actin assembly in the absence or presence...
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