Polycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins, which tag hypoxia-inducible factor (HIF) ␣ subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-␣ stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1 ؊/؊ or Phd3 ؊/؊ mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2␣, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1␣ accumulated in both the kidney and liver. Elevated HIF-1␣ levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2␣/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway. (Blood. 2008; 111:3229-3235)

Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of alpha-smooth muscle actin (alpha-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of alpha-SMA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of alpha-SMA+ cells towards osteoprogenitor lineage. Utilizing alpha-SMAGFP transgene, alpha-SMAGFP+ positive cells were detected in the microvasculature and in the osteoprogenitor population within bone marrow stromal cells. Osteogenic and adipogenic induction stimulated expression of bone and fat markers in the alpha-SMAGFP+ population derived from bone marrow or adipose tissue. In adipose tissue, alpha-SMA+ cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, alpha-SMA+/CD45-/Sca1+ progenitors were highly enriched. Following cell sorting and transplantation of expanded pericyte/myofibroblast populations, donor-derived differentiated osteoblasts and new bone formation was detected. Our results show that cells with a pericyte/myofibroblast phenotype have the potential to differentiate into functional osteoblasts.

The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the treatment of inflammatory disorders. We have previously shown that PDE8 regulates T cell motility. Here, for the first time, we report that PDE8A exerts part of its control of T cell function through the V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase signaling pathway. To examine T cell motility under physiologic conditions, we analyzed T cell interactions with endothelial cells and ligands in flow assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 suppresses adhesion of in vivo myelin oligodendrocyte glycoprotein (MOG) activated inflammatory CD4 + T effector (Teff) cells to brain endothelial cells under shear stress. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP levels around Raf-1 thereby protecting it from protein kinase A (PKA) mediated inhibitory phosphorylation. To test the function of this complex in Teff cells, we used a cell permeable peptide that selectively disrupts the PDE8A-Raf-1 interaction. The disruptor peptide inhibits the Teff-endothelial cell interaction more potently than the enzymatic inhibitor. Furthermore, the LFA-1/ICAM-1 interaction was identified as a target of disruptor peptide mediated reduction of adhesion, spreading and locomotion of Teff cells under flow. Mechanistically, we observed that disruption of the PDE8A-Raf-1 complex profoundly alters Raf-1 signaling in Teff cells. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase complex disruptors decreases Teff cell adhesion and migration under flow, and represents a novel approach to target T cells in inflammation.Keywords: PDE8 CD4 + T cells Integrins Autoimmunity T cell motility, Inflammation 2 IntroductionLigand binding to Gs-coupled receptors leads to the generation of the second messenger cAMP following activation of the enzyme adenylyl cyclase. Stimulation of the T cell antigen receptor (TCR) also leads to elevation of cAMP which is known to inhibit T cell proximal signaling, IL-2 production and T cell proliferation [1,2]. cAMP exerts these inhibitory effects in T cells through cAMP dependent protein kinase (PKA) which blocks the mitogen-activated protein kinase (MAPK) and nuclear factor of activated T cells (NFAT) dependent signaling pathways [3]. The inhibitory action of cAMP is eliminated through the action of phosphodiesterase (PDE) enzymes that hydrolyze cAMP. PDEs 3B, 4A, 4B, 4D, 7A1, 7A3 and 8A1 are the isoforms expressed in T cells [4][5][6][7][8]. V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is an upstream regulator of the MAPK-extracellular signal-regulated kinase (ERK)1/2 module, which controls many fundamental biological processes, including T cell proliferation, survival and adhesion [9][10][11][12]. In this pathway Raf-1 phosphorylates and activates MAP/ERK kinase (MEK)1/2, which in turn phosphorylate and activate ERK1/2. ERK has more than 150 known substrates [9,12], which med...

After decades of development, inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice for the treatment of inflammatory disorders, with three PDE4 inhibitors being in clinical use as therapeutics for psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease and atopic dermatitis. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. We have previously demonstrated a role for the PDE8A-Raf-1 kinase complex in the regulation of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) activated CD4+ effector T cell adhesion and locomotion by a mechanism that differs from PDE4 activity. In this study, we explored the in vivo treatment of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) induced in mice immunized with MOG using the PDE8-selective inhibitor PF-04957325. For treatment in vivo, mice with EAE were either subcutaneously (s.c.) injected three times daily (10 mg/kg/dose), or were implanted subcutaneously with Alzet mini-osmotic pumps to deliver the PDE8 inhibitor (15.5 mg/kg/day). The mice were scored daily for clinical signs of paresis and paralysis which were characteristic of EAE. We observed the suppression of the clinical signs of EAE and a reduction of inflammatory lesion formation in the CNS by histopathological analysis through the determination of the numbers of mononuclear cells isolated from the spinal cord of mice with EAE. The PDE8 inhibitor treatment reduces the accumulation of both encephalitogenic Th1 and Th17 T cells in the CNS. Our study demonstrates the efficacy of targeting PDE8 as a treatment of autoimmune inflammation in vivo by reducing the inflammatory lesion load.

The levels of cAMP are regulated by phosphodiesterases (PDE), which are targets for the treatment of inflammatory disorders. We previously showed that PDE8A regulates T cell motility. We report here that PDE8A exerts its control of T cell function through the Raf-1 kinase signaling pathway. To mimic T cell migration under physiologic conditions, we performed flow chamber assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 significantly suppresses rolling and adhesion of in vivo MOG35-55 activated inflammatory CD4+ T effector (Teff) cells while interacting with inflamed brain endothelial cells under shear flow conditions. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP around Raf-1 thereby protecting it from protein kinase A mediated inhibitory phosphorylation. To test this in Teff cells, we used a cell permeable peptide (DP) that disrupts the PDE8A-Raf-1 signaling complex. Disruption of the PDE8A-Raf-1 complex by DP significantly reduces adhesion of Teff cells to endothelial cells. We further observed that disrupting PDE8A-Raf-1 through DP specifically reduces adhesion, spreading and locomotion of Teff cells while interacting with the vascular adhesion molecule ICAM-1. Our investigation of the effect of PDE8 inhibitor on chronic and relapsing-remitting experimental autoimmune encephalomyelitis (EAE) in vivo indicate suppression of clinical and histopathological signs of disease. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase signaling complex disruptors significantly decreases Teff cell adhesion and migration on endothelial cells, and represents a novel approach to treat autoimmune inflammation in vivo.