The PICALM Protein Plays a Key Role in Iron Homeostasis and Cell Proliferation

<div><p>The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human <em>PICALM</em> gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the <em>PICALM</em> locus to late-onset Alzheimer's disease. Inactivating and hypomorphic <em>Picalm</em> mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM’s function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant <em>fit1</em> mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral <em>PICALM</em> expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.</p> </div

<i>PICALM-deficient</i> MEFs proliferate more rapidly in the presence of iron supplementation and are more sensitive to iron chelation.

<p>(<b>A,B</b>) Representative proliferation curves of non-immortalized <i>Picalm</i>^NULL and WT MEFs without (A) and with (B) 50 µM ferric ammonium citrate (FAC). FAC restores proliferation in <i>Picalm</i>^NULL cells to levels in WT cells. (<b>C,D</b>) Mean cell numbers (+/− standard error) of non-immortalized <i>Picalm</i>^NULL and WT MEFs grown for 5 days without (C) and with (D) 50 µM FAC. N_exp  = 3. *p<0.04 compared with WT cells. (<b>E,F</b>) Representative proliferation curves of immortalized <i>Picalm</i>^NULL MEFs transduced with empty vector (control) or <i>PICALM</i> grown for 4 days in the absence (E) or presence (F) of 2.5 µM deferoxamine (DFO). (<b>G</b>) Mean cell number of <i>Picalm</i>^NULL MEFs untransduced (Null 3T) or transduced with empty vector (control) or PICALM after 4 days of culture in the presence of 2.5 µM DFO treatment relative to number of untreated cells. N_exp  = 4. *p<0.003 compared with PICALM rescued cells.</p

<i>PICALM-deficient</i> cells display increased total TfR protein and mRNA.

<p>(<b>A</b>) Immunoblot of total TfR protein, PICALM, and β-actin from <i>Picalm</i>^NULL (Null), WT, <i>Picalm</i>^NULL control MEFs infected with empty vector (Null + control), or <i>Picalm</i>^NULL MEFs rescued with PICALM (Null + PICALM). <i>PICALM</i> cDNA encodes for the larger isoform. (<b>B</b>) Quantitation of TfR shown in panel A immunoblot normalized to β-actin, with values shown relative to TfR level in WT MEFs. N_exp  = 3. *p<0.04. (<b>C</b>) RT-PCR quantitation of TfR mRNA in <i>PICALM-deficient</i> (Null) MEFs, WT MEFs, <i>Picalm</i>^NULL MEFs transduced with empty vector (Null + Control) or rescued with PICALM (Null + PICALM). Results are normalized to levels in Null MEFs or Null + Control cells. N_exp  = 3. *p<0.04 compared with WT cells.</p

<i>PICALM-deficient</i> MEFs exhibit decreased intracellular iron compared with WT or PICALM rescued MEFs.

<p>(<b>A</b>) Chelation of Phen Green SK (the fluorescence of which inversely correlates with intracellular iron levels) was measured in WT (lines 7T, 20T) or <i>PICALM-deficient</i> (<i>Picalm</i>^NULL – lines 3T, 24T, 29T) MEFs. Values were normalized to those in WT MEFs. N_exp  = 3. *p<0.04 compared with WT. (<b>B</b>) Chelation of Phen Green SK in <i>Picalm</i>^NULL cells that were uninfected or rescued with PICALM. Values are normalized to levels in PICALM-rescued cells. N_exp  = 3. *p<0.04 compared with PICALM-rescued cells. (<b>C</b>) Total intracellular iron levels in <i>Picalm</i>^NULL (line 3T) and PICALM rescued MEFs, normalized to levels in <i>Picalm</i>^NULL cells. N_exp  = 3. MFI: mean fluorescence intensity.</p