The main physiological regulator of erythropoiesis is the hematopoietic growth factor erythropoietin (EPO), which is primarily produced in the kidney of adult mammals and is induced in response to hypoxia. Binding of EPO to the EPO receptor (EPO-R), a member of the cytokine receptor superfamily, controls the terminal maturation of red blood cells. So far, EPO has been reported to act mainly on erythroid precursor cells. However, we have detected mRNA encoding both EPO and EPO-R in mouse brain by reverse transcription-PCR. Exposure to 0.1% carbon monoxide, a procedure that causes functional anemia, resulted in a 20-fold increase of EPO mRNA in mouse brain as quantified by competitive reverse transcription-PCR, whereas the EPO-R mRNA level was not influenced by hypoxia. Binding studies on mouse brain sections revealed defined binding sites for radioiodinated EPO in distinct brain areas. The specificity of EPO binding was assessed by homologous competition with an excess of unlabeled EPO and by using two monoclonal antibodies against human EPO, one inhibitory and the other noninhibitory for binding of EPO to EPO-R. Major EPO binding sites were observed in the hippocampus, capsula interna, cortex, and midbrain areas. Functional expression of the EPO-R and hypoxic upregulation of EPO suggest a role of EPO in the brain.Erythropoietin (EPO) is a 30.4-kDa glycoprotein which represents the major regulator of erythropoiesis (reviewed in refs. 1-3). The main site of EPO production switches during development from the fetal liver to the adult kidney. Fibroblast-like type I interstitial cells have been identified as the EPO-producing cell population in the kidney (4, 5). In the liver, both a subset of hepatocytes and the nonparenchymal Ito cells, also known as fat-storing or perisinusoidal cells, have been reported to be the source of EPO production (6, 7). EPO gene expression is regulated in an oxygen-dependent manner: hypoxic exposure results in elevated EPO production in mammals and in the EPO-producing human hepatoma cell lines HepG2 and Hep3B (1-3). Binding of EPO to the EPO receptor (EPO-R), a member of the cytokine receptor superfamily (8), leads to enhanced red blood cell production by suppressing programmed cell death of erythroid progenitor cells (reviewed in ref. 9). So far, EPO has been shown to act mainly on erythroid cells. However, apart from its key function in erythropoiesis, EPO may also influence the functional behavior of nonerythroid cells: EPO might have mitogenic and chemotactic effects on endothelial cells (10) and fetal liver stromal cells (11), both cell types harboring the EPO-R (12, 11). Stimulation of differentiation and increased DNA synthesis in response to EPO has been observed in megakaryocytes in vitro (13,14), which is consistent with functional expression of EPO-R The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. on those ce...

SummaryBackgroundPhosphorylation of the transcriptional coactivator YAP1 is a key event in defining Hippo signaling outputs. Previous studies demonstrated that phosphorylation of YAP1 at serine 127 (S127) sequesters YAP1 in the cytoplasm and consequently inhibits YAP1 transcriptional activity. Mammalian tissue-culture experiments suggest that downstream of MST1/2 signaling, LATS1/2 function as YAP1-S127 kinases. However, studies of Mst1/2 knockout mouse models revealed that the identity of the physiological YAP1-S127 kinase(s) in certain tissues, such as the intestine, remains unknown.ResultsWe show that mammalian NDR1/2 kinases phosphorylate YAP1 on S127 and thereby negatively regulate YAP1 activity in tissue-cultured cells. By studying NDR1/2-deficient mice, we demonstrate the in vivo relevance of NDR1/2-mediated regulation of YAP1. Specifically, upon loss of NDR1/2 in the intestinal epithelium, endogenous S127 phosphorylation is decreased whereas total YAP1 levels are increased. Significantly, ablation of NDR1/2 from the intestinal epithelium renders mice exquisitely sensitive to chemically induced colon carcinogenesis. Analysis of human colon cancer samples further revealed that NDR2 and YAP1 protein expression are inversely correlated in the majority of samples with high YAP1 expression. Collectively, we report NDR1/2 as physiological YAP1-S127 kinases that might function as tumor suppressors upstream of YAP1 in human colorectal cancer.ConclusionsWe establish mammalian NDR1/2 as bona fide kinases that target YAP1 on S127 in vitro and in vivo. Our findings therefore have important implications for a broad range of research efforts aimed at decoding and eventually manipulating YAP1 biology in cancer settings, regenerative medicine, and possibly also noncancer human diseases.

Blastocyst-derived pluripotent mouse embryonic stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or 1% (hypoxia) oxygen during the first 5 days of differentiation. Microelectrode measurements of pericellular oxygen tension within 13-to 14-day-old EBs (diameter jtm) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal (10 ,ag) was denatured in formamide/formaldehyde and electrophoresed through a 1% agarose gel containing 6% formaldehyde as described (35). After pressure blotting (PosiBlot, Stratagene) to a nylon membrane (Biodyne A, Pall), followed by UV crosslinking (Stratalinker, Stratagene), the RNA was hybridized to 32P-labeled probes according to the manufacturer's directions (Pall). The radioactive signal was recorded and quantified by using a PhosphorImager (Molecular Dynamics). The aldolase A probe was cloned from a HepG2 cDNA library and the 3-actin cDNA was purchased from Clontech. Aldolase A and VEGF mRNA signals were normalized to the ,3-actin signal to correct for loading and blotting differences. The mouse VEGF probe was derived from a 644-bp PCR fragment generated by using the forward primer A (5'-GCGGGCTGCCTCGCAGTC-3') and the reverse primer B (5'-TCACCGCCTTGGCTTGTCAC-3'), corresponding to bp 16-33 and 659-690, respectively, of the murine VEGF cDNA sequence according to ref. 36. The internal primer C (5'-GCTCACAGTGATTTTCTGGCT-3') corresponding to bp 496-516 (36) was designed to exclusively amplify the cDNA of transcripts encoding the 164 amino acid isoform of VEGF. The competitor VEGF template was constructed exactly as described by Siebert and Larrick (37), and competitive PCR was performed using the conditions reported recently for EPO (38). (1996) Proc. Natl. Acad. Sci. USA 93 (1996) 2869 during further development, 13-to 14-day-old EBs (n = 9) were incubated with calcein AM. Fig. 1C shows an example of one EB mechanically sectioned in the equatorial plane since penetration of the scanning laser is limited to a depth of about 250 ,m (47). As in Fig. 1B RESULTS Differentiation of ES Cells in

Abstract-The Ca 2؉ -binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca 2؉ -buffering in specific cells including neurons and have profound effectson spatiotemporal aspects of Ca 2؉ transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV؊/؊ mice is viewed as a specific compensation mechanism to maintain Ca 2؉ homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca 2؉ buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV؊/؊ PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 m width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 m thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB؊/؊ mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca 2؉ homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca 2؉ signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca 2؉ fluxes.Key words: calcium-binding, buffers, EF-hand, homeostasis, morphology.Ca 2ϩ ions are such ubiquitous second messengers that meaningful information must be contained in the subtle spatiotemporal aspects of Ca 2ϩ transients. A complex machinery of Ca 2ϩ entry and release systems, mobile and immobile Ca 2ϩ buffers, transient Ca 2ϩ -storage devices and Ca 2ϩ -extrusion systems governs the shape and spreading of intracellular Ca 2ϩ transients (Berridge et al., 2003). Affinities, kinetics of binding and release of Ca 2ϩ ions, the relative mobility and the geometrical distribution of all components, that is, the interplay between these systems finally shapes the spatiotemporal aspects of a Ca 2ϩ signal. Cerebellar Purkinje cells (PC) are characterized by extensive Ca 2ϩ signaling in somata, dendrites and spines elicited by either climbing fiber or parallel fiber stimulation. Following depolarization-evoked rises in the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) in the PC somata, endoplasmic reticulum (ER) and plasma membrane Ca 2ϩ pumps and the Na ϩ -Ca 2ϩ exchanger contribute to PC [Ca 2ϩ ] i clearance (Fierro et al., 1998). Since these systems only accounted for approximately 60% of total Ca 2ϩ clearing, mitochondria were additionally postulated to play a role. These organelles have...