The Cellular
Trafficking of the Secretory Proprotein Convertase PCSK9 and Its Dependence
on the LDLR. ·
Nassoury N, Blasiole DA, Tebon Oler A,
Benjannet S, Hamelin J, Poupon V, McPherson PS, Attie AD, Prat A, Seidah NG. Laboratory
of Biochemical Neuroendocrinology, Clinical
Research Mutations
in the proprotein convertase
PCSK9 gene are associated with autosomal dominant
familial hyper- or hypocholesterolemia. These
phenotypes are caused by a gain or loss of function of proprotein
convertase subtilisin kexin 9 (PCSK9) to elicit the degradation of the
low-density lipoprotein receptor (LDLR) protein. Herein, we asked whether
the subcellular localization of wild-type PCSK9
or mutants of PCSK9 and the LDLR would provide insight into the mechanism
of PCSK9-dependent LDLR degradation. We show that the LDLR is the dominant
partner in regulating the cellular trafficking of PCSK9. In cells lacking the
LDLR, PCSK9 localized in the endoplasmic reticulum (ER). In cells
expressing the LDLR, PCSK9 sorted to post-ER compartments (i.e. endosomes in cell lines and Golgi
apparatus in primary hepatocytes), where it colocalized with the LDLR. In cell lines, PCSK9 also colocalized with the LDLR at the cell surface,
requiring the presence of the C-terminal Cys/His-rich
domain of PCSK9. We provide evidence that PCSK9 promotes the degradation of
the LDLR by an endocytic mechanism, as small
interfering RNA-mediated knockdown of the clathrin
heavy chain reduced the functional activity of PCSK9. We also compared the subcellular localization of natural mutants of PCSK9
with that of the wild-type enzyme in human hepatic (HuH7) cells. Whereas
the mutants associated with hypercholesterolemia (S127R, F216L and R218S)
localized to endosomes/lysosomes, those
associated with hypocholesterolemia did not reach
this compartment. We conclude that the sorting of PCSK9 to the cell surface
and endosomes is required for PCSK9 to fully promote
LDLR degradation and that retention in the ER prevents this activity.
Mutations that affect this transport can lead to hyper- or hypocholesterolemia. PMID: 17461796
[PubMed - as supplied by publisher] Ann
Endocrinol (Paris). 2007 Mar 26; [Epub ahead of print] [After the LDL
receptor and apolipoprotein B, autosomal dominant hypercholesterolemia reveals its
third protagonist: PCSK9.] [Article in
French] ·
Abifadel M, Rabes JP, Boileau C, Varret M. Inserm, U781, Paris,
France; APHP, hopital Necker-Enfants
malades, 149, rue de Sevres, 75743 Paris, France;
faculte de medecine, universite Paris-V, Paris, France; Faculte
de pharmacie, universite
Saint-Joseph, BP 11, 5076 Beyrouth, Liban. The
genes encoding the low-density lipoproteins receptor and its ligand apolipoprotein B, have been the only two genes classically implicated
in autosomal dominant hypercholesterolemia. We
have identified in 2003, the third gene implicated in this disease: PCSK9 (Proprotein Convertase Subtilin Kexin 9). Several
mutations (p.S127R, p.F216L, p.D374Y...) of this gene have been reported to
cause hypercholesterolemia by a gain of function leading to a reduction of
LDL receptor levels. Other variations of PCSK9 are conversely associated
with hypocholesterolemia particularly the
non-sense p.Y142X and p.C679X mutations found in 2% of black Americans and
associated with a decrease of LDL levels and coronary heart diseases. PCSK9
substrates and exact role have not been elucidated yet, but it seems that
PCSK9 is definitely a major actor in cholesterol homeostasis. PCSK9
inhibitors might constitute new therapeutic targets that would decrease
plasma LDL cholesterol levels and be synergistic with statin
drugs. PMID: 17391637
[PubMed - as supplied by publisher Atherosclerosis. 2007 Feb 20; [Epub
ahead of print] Genetic
variants in PCSK9 in the Japanese population: Rare genetic variants in
PCSK9 might collectively contribute to plasma LDL cholesterol levels in the
general population. ·
Miyake Y, Kimura R, Kokubo Y, Okayama A, Tomoike H, Yamamura T, Miyata T. Department of
Etiology and Pathophysiology, National
Cardiovascular Center Research Institute, The aim
of this study was to investigate whether plasma low-density lipoprotein
cholesterol (LDL-C) levels in the general population are influenced by rare
sequence variations in the PCSK9 gene. We sequenced the promoter and coding
regions of the PCSK9 gene in individuals from the general population
(n=3655) with the lowest (n=78) and highest (n=96) LDL-C levels and in
individuals taking antihypercholesterolemia
medication (n=96). We identified 33 sequence variants in the PCSK9 gene
among which 24 were specific for Japanese. Statistical analysis showed that
one missense mutation, R93C, was associated with
low LDL-C levels. The other variants had no association with LDL-C levels
or the numbers of individuals with the variants were too small for
statistical analysis. A comparison of the numbers of individuals with nonsynonymous mutations between the low LDL-C and high
LDL-C/treatment groups found that four missense
mutations and one nonsense mutation were identified only in the low LDL-C
group and six missense mutations were identified
only in the high LDL-C/treatment group. As we have analyzed groups at
opposite ends of the LDL-C spectrum, it is likely that some of these nonsynonymous mutations may be associated with either
low or high LDL-C in the Japanese population. Based on the extremely high
frequencies of the nonsynonymous mutations in
PCSK9 compared with those of LDLR or apoB-100, PCSK9 mutations could be
important factors that cumulatively influence plasma LDL-C levels in the
general population. PMID: 17316651
[PubMed - as supplied by publisher] Chronic Immune Therapy Induces a Progressive Increase in Intratumoral T Suppressor Activity and a Concurrent
Loss of Tumor-Specific CD8+ T Effectors in her-2/neu Transgenic Mice
Bearing Advanced Spontaneous Tumors. Lab Invest.
2006 May 15; [Epub ahead of print] J
Bone Miner Res. 2006 Jun;21(6):946-55.
Links 
Links
Nair
RE, Kilinc MO, Jones
SA, Egilmez NK.
A single intratumoral injection of IL-12 and
GM-CSF-encapsulated microspheres induces the
complete regression of advanced spontaneous tumors in her-2/neu transgenic
mice. However, tumor regression in this model is transient and long-term
cure is not achieved due to recurrence. Posttherapy
molecular analysis of immune activation/suppression markers within the
tumor microenvironment demonstrated a dramatic up-regulation of IFN-gamma
and a concomitant down-regulation of Forkhead/winged-helix
protein 3 (Foxp3), TGFbeta, and IL-10 expression.
Therapy-induced reversion of immune suppression was transient since all
three markers of suppression recovered rapidly and surpassed pretherapy levels by day 7 after treatment, resulting
in tumor resurgence. Repeated treatment enhanced short-term tumor
regression, but did not augment long-term survival. Serial long-term
analysis demonstrated that although chronic stimulation enhanced the
IFN-gamma response, this was countered by a parallel increase in Foxp3, TGFbeta, and IL-10 expression. Analysis of
tumor-infiltrating T lymphocyte populations showed that the expression of
Foxp3 and IL-10 was associated with CD4(+)CD25(+)
T cells. Repeated treatment resulted in a progressive increase in
tumor-infiltrating CD4(+)CD25(+)Foxp3(+) T
suppressor cells establishing their role in long-term neutralization of antitumor activity. Analysis of tumor-infiltrating CD8(+) T cells demonstrated that although treatment
enhanced IFN-gamma production, antitumor cytotoxicity was diminished. Monitoring of CD8(+) T cells that specifically recognized a dominant
MHC class I her-2/neu peptide showed a dramatic increase in
tetramer-specific CD8(+) T cells after the first treatment; however,
continuous therapy resulted in the loss of this population. These results
demonstrate that both enhanced suppressor activity and deletion of
tumor-specific T cells are responsible for the progressive loss of efficacy
that is associated with chronic immune therapy.
PMID: 16751376 [PubMed - in process]
UBD, a downstream element of FOXP3, allows the
identification of LGALS3, a new marker of human regulatory T cells.
Ocklenburg F, Moharregh-Khiabani D, Geffers R, Janke V, Pfoertner S, Garritsen H, Groebe L, Klempnauer J, Dittmar KE, Weiss
S, Buer J, Probst-Kepper M.
1Junior Research Group for Xenotransplantation,
Department of Visceral and Transplant Surgery,
Here, we report the identification of the ubiquitin-like
gene UBD as a downstream element of FOXP3 in human activated regulatory CD4(+)CD25(hi) T cells (T(reg)).
Retroviral transduction of UBD in human allo-reactive
effector CD4(+) T helper
(T(h)) cells upregulates CD25 and mediates downregulation of IL4 and IL5 expression similar to overexpression of FOXP3. Moreover, UBD impairs T(h) cell proliferation without upregulation
of FOXP3 and impairs calcium mobilization. In the presence of ionomycin, overexpression of
UBD in T(h) cells leads to the induction of IL1R2
that resemble FOXP3-transduced T(h) cells and naturally derived T(reg) cells. A comparison of the transcriptome
of FOXP3- and UBD-transduced T(h)
cells with T(reg) cells allowed the
identification of the gene LGALS3. However, high levels of LGALS3 protein
expression were observed only in human CD4(+)CD25(hi)
derived T(reg) cells and FOXP3-transduced T(h)
cells, whereas little was induced in UBD-transduced
T(h) cells. Thus, UBD contributes to the anergic
phenotype of human regulatory T cells and acts downstream in FOXP3 induced
regulatory signaling pathways, including regulation of LGALS3 expression.
High levels of LGALS3 expression represent a FOXP3-signature of human
antigen-stimulated CD4(+)CD25(hi) derived regulatory T cells.Laboratory
Investigation advance online publication, 15 May 2006;
doi:10.1038/labinvest.3700432.
PMID: 16702978 [PubMed - as supplied by
publisher]
Fibroblast growth factor-2 is an
immediate-early gene induced by mechanical stress in osteogenic
cells.
Li CF, Hughes-Fulford M.
Laboratory of Cell Growth, Northern California Institute for Research and
Education, San Francisco, California, USA.
Fifteen minutes of physiological MS induces FGF-2 in osteogenic
cells. Here, we show that MS induced proliferation in both MC3T3-E1 and BMOp cells isolated from Fgf2(+/+)
mice; Fgf2(-/-) BMOp cells required exogenous
FGF-2 for a normal proliferation response. The induction of fgf-2 is
mediated by PKA and ERK pathways. INTRODUCTION: Mechanical stress (MS)
induces gene expression and proliferation of osteogenic
MC3T3-E1 cells. We have previously shown that physiological levels of MS in
MC3T3-E1 cells causes extracellular
signal-regulated kinase (ERK)1/2
phosphorylation. Here we evaluate the induction
and importance of fibroblast growth factor-2 (FGF-2) for MS-induced proliferation.
MATERIALS AND METHODS: We characterized the MS induction of fgf-2 using a
15-minute pulse of 120 mustrain and studied the
stability of fgf-2 message using actinomycin D.
Bone marrow stromal cells (BMOp)
isolated from Fgf2(-/-) and Fgf2(+/+) mice were used to study the
importance of FGF-2 in MS-induced proliferation. RESULTS: We found that the
induction of fgf-2 by MS is dependent on both protein kinase
A (PKA) and ERK pathways. MS transiently induces fgf-2 within 30 minutes.
FGF-2 receptor (FGFR2) was also significantly increased within 1 h. All
three isoforms of FGF-2 (24, 22, and 18 kDa) were significantly increased by MS. The
MS-mediated increase of fgf-2 mRNA was caused by new synthesis and not
stabilization. Pretreatment of MC3T3-E1 cells with cycloheximide
showed that the induction of fgf-2 did not require new protein synthesis. Pretreating MC3T3-E1 cells with the mitogen-activated
protein kinase (MAPK)/ERK kinase
1/2 (MEK1/2) inhibitor, U0126, or H-89, a PKA inhibitor, significantly
inhibited the induction of fgf-2, showing that mechanical induction of
fgf-2 is dependent on ERK and PKA signaling pathways. The downstream
consequence of a single 15-minute stress pulse was a 3.5-fold increase in
cell number in MC3T3-E1 compared with growth in nonstressed
control cells. In studies using bone marrow osteoprogenitor
cells (BMOp) isolated from Fgf2(+/+)and Fgf2(-/-)
mice, we found that FGF-2 was necessary for a full proliferative
response to MS. CONCLUSIONS: These studies show that FGF-2 is an immediate-early
gene induced by MS, and its expression is mediated by both the PKA and MAPK
signal transduction pathways. FGF-2 was required for a full proliferative response.
PMID: 16753025 [PubMed - in process]