Identification of a Novel Function of PiT1 Critical for Cell Proliferation and Independent of Its Phosphate Transport Activity

PiT1 is a Na⁺-phosphate (P_i) cotransporter located at the plasma membrane that enables P_i entry into the cell. Its broad tissue expression pattern has led to the idea that together with the closely related family member PiT2, PiT1 is the ubiquitous supplier of P_i to the cell. Moreover, the role of P_i in phosphorylation reactions, ATP production, DNA structure, and synthesis has led to the view that P_i availability could be an important determinant of cell growth. However, these issues have not been clearly addressed to date, and the role of either P_i or PiT proteins in cell proliferation is unknown. Using RNA interference in HeLa and HepG2 cells, we show that transient or stable PiT1 depletion markedly reduces cell proliferation, delays cell cycle, and impairs mitosis and cytokinesis. In vivo, PiT1 depletion greatly reduced tumor growth when engineered HeLa cells were injected into nude mice. We provide evidence that this effect on cell proliferation is specific to PiT1 and not shared by PiT2 and is not the consequence of impaired membrane Na⁺-P_i transport. Moreover, we show that modulation of cell proliferation by PiT1 is independent from its transport function because the proliferation of PiT1-depleted cells can be rescued by non-transporting PiT1 mutants. PiT1 depletion leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase, whereas other MAP kinases and downstream targets of mammalian target of rapamycin (mTOR) remain unaffected. This study is the first to describe the effects of a P_i transporter in cell proliferation, tumor growth, and cell signaling.     

The role of the death-domain kinase RIP in tumour-necrosis-factor-induced activation of mitogen-activated protein kinases

The death-domain kinase RIP (receptor-interacting protein) is an important effector of tumour necrosis factor (TNF) signalling and is essential for TNF-induced nuclear factor-κB activation. However, the function of RIP in the TNF-induced activation of mitogen-activated protein kinases (MAPKs) has not been fully investigated. In this report, using Rip null (Rip^−/−) mouse fibroblast cells, we investigated whether RIP is required for TNF-induced activation of the MAPKs extracellular-signal-related kinase (ERK), p38 and c-Jun amino-terminal kinase (JNK). We found that TNF-induced activation of ERK, p38 and JNK is decreased in Rip^−/− cells. The activation of these kinases by interleukin-1 is normal in Rip^−/− cells. More importantly, we showed that the kinase activity of RIP is needed for ERK activation.     

ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis

Apoptosis signal-regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H₂O₂, and activates c-Jun NH₂-terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF- and H₂O₂-induced sustained activations of JNK and p38 are lost in ASK1^–/– embryonic fibroblasts, and that ASK1^–/– cells are resistant to TNF- and H₂O₂-induced apoptosis. TNF- but not Fas-induced apoptosis requires ROS-dependent activation of ASK1–JNK/p38 pathways. Thus, ASK1 is selectively required for TNF- and oxidative stress-induced sustained activations of JNK/p38 and apoptosis.     

JNK2 mediates TNF-induced cell death in mouse embryonic fibroblasts via regulation of both caspase and cathepsin protease pathways

Recent studies strongly suggest an active involvement of the c-Jun N-terminal kinase (JNK) signaling pathway in tumor necrosis factor (TNF)-induced apoptosis. The direct evidence for the role of JNK and its isoforms has been missing and the mechanism of how JNK actually could facilitate this process has remained unclear. In this study, we show that Jnk2-/- primary mouse embryonic fibroblasts (pMEFs) exhibit resistance towards TNF-induced apoptosis as compared to corresponding wild-type and Jnk1-/- pMEFs. JNK2-deficient pMEFs could be resensitized to TNF via retroviral transduction of any of the four different JNK2 splicing variants. Jnk2-/- pMEFs displayed deficient and delayed effector caspase activation as well as impaired cytosolic cystein cathepsin activity: processes that both were needed for efficient TNF-induced apoptosis in pMEFs. Our work demonstrates that JNK has a central role in the promotion of TNF-induced apoptosis in pMEFs, and that the JNK2 isoform can regulate both mitochondrial and lysosomal death pathways in these cells.     

Genetic inactivation of Par4 results in hyperactivation of NF-kappaB and impairment of JNK and p38

The Par4 gene was first identified in prostate cells undergoing apoptosis after androgen withdrawal. PAR4 was subsequently shown to interact with, and inhibit, atypical protein kinase C isoforms, functioning as a negative regulator of the NF-κB pathway. This may explain its pro-apoptotic function in overexpression experiments. To determine the physiological role of PAR4, we have derived primary embryonic fibroblasts (EFs) from Par4^−/− mice. We show here that loss of PAR4 leads to a reduction in the ability of tumour necrosis factor-α (TNF-α) to induce apoptosis by increased activation of NF-κB. Consistent with recent reports demonstrating the antagonistic actions of NF-κB and c-Jun amino-terminal kinase (JNK) signalling, we have found that Par4^−/− cells show a reduced activation of the sustained phase of JNK and p38 stimulation by TNF-α and interleukin 1. Higher levels of an anti-apoptotic JNK-inhibitor protein, X-chromosome-linked inhibitor of apoptosis, in Par4^−/− EFs might explain the inhibition of JNK activation in these cells.     

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

The general phosphate need in mammalian cells is accommodated by members of the P_i transport (PiT) family (SLC20), which use either Na⁺ or H⁺ to mediate inorganic phosphate (P_i) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na⁺-dependent P_i (NaP_i) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with ³²P_i as a traceable P_i source. For PiT1, the Michaelis-Menten constant for P_i was determined as 322.5 ± 124.5 µM. PiT2 was analyzed for the first time and showed positive cooperativity in P_i uptake with a half-maximal activity constant for P_i of 163.5 ± 39.8 µM. PiT1- and PiT2-mediated Na⁺-dependent P_i uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na⁺ dependency patterns. However, only PiT2 was capable of Na⁺-independent P_i transport at acidic pH. Study of the impact of divalent cations Ca²⁺ and Mg²⁺ revealed that Ca²⁺ was important, but not critical, for NaP_i transport function of PiT proteins. To gain insight into the NaP_i cotransport function, we analyzed PiT2 and a PiT2 P_i transport knockout mutant using ²²Na⁺ as a traceable Na⁺ source. Na⁺ was transported by PiT2 even without P_i in the uptake medium and also when P_i transport function was knocked out. This is the first time decoupling of P_i from Na⁺ transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E⁵⁵ and E⁵⁷⁵ are responsible for linking P_i import to Na⁺ transport in PiT2. inorganic phosphate transport; retroviral receptor; SLC20     

Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha

We used a gene knockout approach to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NF-κB pathways downstream of TNF-α in the context of α(2) type I collagen gene (COL1A2) expression. In JNK₁^−/−-JNK₂^−/− (JNK^−/−) fibroblasts, TNF-α inhibited basal COL1A2 expression but had no effect on TGF-β-driven gene transactivation unless jnk1 was introduced ectopically. Conversely, in NF-κB essential modulator^−/− (NEMO^−/−) fibroblasts, lack of NF-κB activation did not influence the antagonism exerted by TNF-α against TGF-β but prevented repression of basal COL1A2 gene expression. Similar regulatory mechanisms take place in dermal fibroblasts, as evidenced using transfected dominant-negative forms of MKK4 and IKK-α, critical kinases upstream of the JNK and NF-κB pathways, respectively. These results represent the first demonstration of an alternate usage of distinct signaling pathways by TNF-α to inhibit the expression of a given gene, COL1A2, depending on its activation state.     

Vitamin K2 Biosynthetic Enzyme,UBIAD1 Is Essential for Embryonic Development of Mice

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K₂ biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1 ^−/−) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1 ^−/− embryonic stem (ES) cells failed to synthesize vitamin K₂ but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1 ^+/− mice developed normally, exhibiting normal growth and fertility. Vitamin K₂ tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K₂ synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1 ^+/− E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1 ^−/− mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1 ^+/− mice. These results suggest that UBIAD1 is responsible for vitamin K₂ synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K₂, but may have additional functions beyond the biosynthesis of vitamin K₂.     

Differential Regulation of c-Jun Protein Plays an Instrumental Role in Chemoresistance of Cancer Cells

The chemotherapeutic drug cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) is widely used in the treatment of human cancers. However, the mechanism underlying intrinsic tumor resistance to CDDP remains elusive. Here, we demonstrate that treatment with CDDP resulted in down-regulation of c-Jun expression via caspase-9-dependent cleavage of c-Jun at Asp-65 and MEKK1-mediated ubiquitylation and degradation of c-Jun in CDDP-sensitive cancer cells. In contrast, activation of JNK2 (but not JNK1) phosphorylated and up-regulated the expression of c-Jun in CDDP-resistant cells. Activated c-Jun bound to the promoter regions of the MDR1 gene and promoted the expression of MDR1. Expression of a cleavage-resistant c-Jun mutant (D65A) suppressed CDDP-induced apoptosis of CDDP-sensitive cells, whereas depletion of JNK2, c-Jun, or MDR1 in CDDP-resistant cancer cells promoted apoptosis upon CDDP treatment. In addition, mammary gland tumors induced by polyomavirus middle T antigen in JNK2^−/− mice were more sensitive to CDDP compared with those in JNK2^+/+ mice. These findings highlight the instrumental role of c-Jun in the resistance of tumors to treatment with CDDP and indicate that c-Jun is a molecular target for improving cancer therapy.     

TNF-induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members

TNF receptor 1 signaling induces NF-κB activation and necroptosis in L929 cells. We previously reported that cellular inhibitor of apoptosis protein-mediated receptor-interacting protein 1 (RIP1) ubiquitination acts as a cytoprotective mechanism, whereas knockdown of cylindromatosis, a RIP1-deubiquitinating enzyme, protects against tumor necrosis factor (TNF)-induced necroptosis. We report here that RIP1 is a crucial mediator of canonical NF-κB activation in L929 cells, therefore questioning the relative cytoprotective contribution of RIP1 ubiquitination versus canonical NF-κB activation. We found that attenuated NF-κB activation has no impact on TNF-induced necroptosis. However, we identified A20 and linear ubiquitin chain assembly complex as negative regulators of necroptosis. Unexpectedly, and in contrast to RIP3, we also found that knockdown of RIP1 did not block TNF cytotoxicity. Cell death typing revealed that RIP1-depleted cells switch from necroptotic to apoptotic death, indicating that RIP1 can also suppress apoptosis in L929 cells. Inversely, we observed that Fas-associated protein via a death domain, cellular FLICE inhibitory protein and caspase-8, which are all involved in the initiation of apoptosis, counteract necroptosis induction. Finally, we also report RIP1-independent but RIP3-mediated necroptosis in the context of TNF signaling in particular conditions.     

CAPNS1 Regulates USP1 Stability and Maintenance of Genome Integrity

Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (μ-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines. USP1 modulates DNA replication polymerase choice and repair by deubiquitinating PCNA. The ubiquitinated form of the USP1 substrate PCNA is stabilized in CAPNS1-depleted U2OS cells and mouse embryonic fibroblasts (MEFs), favoring polymerase-η loading on chromatin and increased mutagenesis. USP1 degradation directed by the cell cycle regulator APC/C^cdh1, which marks USP1 for destruction in the G₁ phase, is upregulated in CAPNS1-depleted cells. USP1 stability can be rescued upon forced expression of calpain-activated Cdk5/p25, previously reported as a cdh1 repressor. These data suggest that calpain stabilizes USP1 by activating Cdk5, which in turn inhibits cdh1 and, consequently, USP1 degradation. Altogether these findings point to a connection between the calpain system and the ubiquitin pathway in the regulation of DNA damage response and place calpain at the interface between cell cycle modulation and DNA repair.     

Regulation of the Epithelial Adhesion Molecule CEACAM1 Is Important for Palate Formation

Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at various developmental stages before, during, and after palate fusion using GeneChip® microarrays. Ceacam1 was one of the highly up-regulated genes during palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was present in prefusion palatal epithelium and was degraded during fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1 ^−/−) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1 ^−/− mice. TGFβ3 expression, apoptosis, and cell proliferation in palatal epithelium were not affected in the palate of Ceacam1^−/−mice. However, CEACAM1 expression was retained in the remaining MEE of TGFβ-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion.     

The Phosphate Transporter PiT1 (Slc20a1) Revealed As a New Essential Gene for Mouse Liver Development

Background

PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na⁺-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.

Methodology/Principal Findings

To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and null PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and null alleles in heterozygous compounds results in late embryonic lethality (E14.5–E16.5) with phenotypic features intermediate between null and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.

Conclusion/Significance

This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.     

Polyubiquitinated Tristetraprolin Protects from TNF-induced,Caspase-mediated Apoptosis

Binding of TNF to its receptor (TNFR1) elicits the spatiotemporal assembly of two signaling complexes that coordinate the balance between cell survival and cell death. We have shown previously that, following TNF treatment, the mRNA decay protein tristetraprolin (TTP) is Lys-63-polyubiquitinated by TNF receptor-associated factor 2 (TRAF2), suggesting a regulatory role in TNFR signaling. Here we demonstrate that TTP interacts with TNFR1 in a TRAF2-dependent manner, thereby initiating the MEKK1/MKK4-dependent activation of JNK activities. This regulatory function toward JNK activation but not NF-κB activation depends on lysine 105 of TTP, which we identified as the corresponding TRAF2 ubiquitination site. Disabling TTP polyubiquitination results in enhanced TNF-induced apoptosis in cervical cancer cells. Together, we uncover a novel aspect of TNFR1 signaling where TTP, in alliance with TRAF2, acts as a balancer of JNK-mediated cell survival versus death.     

Hsp72 and stress kinase c-jun N-terminal kinase regulate the bid-dependent pathway in tumor necrosis factor-induced apoptosis

The major inducible heat shock protein Hsp72 has been shown to protect cells from certain apoptotic stimuli. Here we investigated the mechanism of Hsp72-mediated protection from tumor necrosis factor (TNF)-induced apoptosis of primary culture of IMR90 human fibroblasts. Hsp72 temporarily blocked apoptosis in response to TNF and permanently protected cells from heat shock. An Hsp72 mutant (Hsp72 Delta EEVD) with a deletion of the four C-terminal amino acids, which are essential for the chaperone function, blocked TNF-induced apoptosis in a manner similar to that of normal Hsp72 but did not inhibit heat shock-induced death. Therefore, the chaperone activity of Hsp72 is dispensable for suppression of TNF-induced apoptosis but is required for protection from heat shock. In fibroblasts derived from Bid knockout mice, similar temporal inhibition of TNF-induced apoptosis was seen. In these cells neither normal Hsp72 nor Hsp72 Delta EEVD conferred additional protection from apoptosis, suggesting that Hsp72 specifically affects Bid-dependent but not Bid-independent apoptotic pathways. Furthermore, both normal Hsp72 and Delta Hsp72EEVD inhibited Bid activation and downstream events, including release of cytochrome c, activation of caspase 3, and cleavage of poly-ADP-ribose polymerase. Both Hsp72 and Delta Hsp72EEVD blocked activation of the stress kinase c-jun N-terminal kinase (JNK) by TNF, and specific inhibition of JNK similarly temporarily blocked Bid activation and the downstream apoptotic events. These data strongly suggest that in TNF-induced apoptosis, Hsp72 specifically interferes with the Bid-dependent apoptotic pathway via inhibition of JNK.     

Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P_i). To better understand phosphorus movement between the bacteroid and the host plant, P_i transport was characterized in R. tropici. We observed two P_i transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K_m and V_max values for the low-affinity system were estimated to be 34 ± 3 μM P_i and 118 ± 8 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively, and the K_m and V_max values for the high-affinity system were 0.45 ± 0.01 μM P_i and 86 ± 5 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively. Both systems were inducible by P_i starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P_i transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the P_i transport rate was correlated with the intracellular ATP concentration. Also, P_i movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P_i transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.