Interfacing protein lysine acetylation and protein phosphorylation: ?Ancient modifications meet on ancient proteins

Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature.     

TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion

Recent studies implicate a role for cell mechanics in cancer progression. The epithelial-to-mesenchymal transition (EMT) regulates the detachment of cancer cells from the epithelium and facilitates their invasion into stromal tissue. Although classic EMT hallmarks include loss of cell–cell adhesions, morphology changes, and increased invasion capacity, little is known about the associated mechanical changes. Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response. Here we demonstrate that transforming growth factor β (TGF-β)–induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins. We find that suppression of the RhoA guanine nucleotide exchange factors (GEFs) LARG and GEF-H1 through TGF-β/ALK5–enhanced proteasomal degradation mediates these changes in cell mechanics and affects EMT-associated invasion. Taken together, our results reveal a functional connection between attenuated stiffness and stiffening response and the increased invasion capacity acquired after TGF-β–induced EMT.     

Exposure of vital cells to necrotic cell lysates induce the IRE1α branch of the unfolded protein response and cell proliferation

Necrosis is a form of cell death that is detrimental to the affected tissue because the cell ruptures and releases its content (reactive oxygen species among others) into the extracellular space. Clusterin (CLU), a cytoprotective extracellular chaperone has been shown to be upregulated in the face of necrosis. We here show that in addition to CLU upregulation, necrotic cell lysates induce JNK/SAPK signaling, the IRE1α branch of the unfolded protein response (UPR), the MAPK/ERK1/2, and the mTOR signaling pathways and results in an enhanced proliferation of the vital surrounding cells. We name this novel response mechanism: Necrosis-induced Proliferation (NiP).     

ING1b negatively regulates HIF1α protein levels in adipose-derived stromal cells by a SUMOylation-dependent mechanism

Hypoxic niches help maintain mesenchymal stromal cell properties, and their amplification under hypoxia sustains their immature state. However, how MSCs maintain their genomic integrity in this context remains elusive, since hypoxia may prevent proper DNA repair by downregulating expression of BRCA1 and RAD51. Here, we find that the ING1b tumor suppressor accumulates in adipose-derived stromal cells (ADSCs) upon genotoxic stress, owing to SUMOylation on K193 that is mediated by the E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4). We demonstrate that ING1b finely regulates the hypoxic response by triggering HIF1α proteasomal degradation. On the contrary, when mutated on its SUMOylation site, ING1b failed to efficiently decrease HIF1α levels. Consistently, we observed that the adipocyte differentiation, generally described to be downregulated by hypoxia, was highly dependent on ING1b expression, during the early days of this process. Accordingly, contrary to what was observed with HIF1α, the absence of ING1b impeded the adipogenic induction under hypoxic conditions. These data indicate that ING1b contributes to adipogenic induction in adipose-derived stromal cells, and thus hinders the phenotype maintenance of ADSCs.Human mesenchymal stem/stromal cells (MSCs) are able to self-renew and differentiate into various cell types. Recently, MSCs have been developed as tools for tissue engineering and cell-based therapies¹ in particular owing to their trophic and immunosuppressive activities.² Conventionally, the bone marrow MSCs (BM-MSCs) and the adipose-derived stem/stromal cells (ADSCs) have constituted the main sources of MSCs for clinical use. These cells are expanded in vitro prior to their application; however, this long-term culture may allow the emergence of senescence and phenotypic alterations, rendering MSCs unsuitable for clinical purposes.³To overcome these issues, MSC culture in conditions mimicking hypoxic niches has been tested.⁴ Low O₂ tensions promote MSC growth, survival and maintain their self-renewing multipotent state.⁵ However, how hypoxia (1% O₂) affects MSC behavior is unclear. Responses to hypoxia are mainly mediated by hypoxia inducible factors (HIFs). HIF1, 2 and 3α subunits, are constitutively degraded in normoxia and stabilized in hypoxia. Consequently, when stabilized they can dimerize with HIF1β, and then translocate into the nucleus to modulate the expression of selected genes. HIF1α is highly expressed in MSCs, controls their metabolic fate and maintains them in an undifferentiated state.⁶ HIF1α has also been shown to delay the occurrence of senescence in MSCs, by repressing E2A and p21 expression.⁷The inhibitors of growth (ING) family genes act as readers of the epigenetic histone code. Among them, ING1 has been described as a type II tumor suppressor, regulating cell growth, DNA repair, apoptosis, chromatin remodeling and senescence.⁸ To some extent, ING1 and HIF might have opposite effects, (e.g. on tumor progression). Indeed, HIF1α, unlike ING1 that inhibits angiogenesis, promotes angiogenesis.⁹ Furthermore, p53, a well-known ING1b interactor, and HIF1α have been shown in several studies to have antagonistic effects. Following DNA damage, p53 induces apoptosis and inhibits survival of cells by reducing activity and levels of HIF1α.^{10, 11}So far, ING4 has been shown as the only ING protein to regulate the hypoxic response. Indeed, by interacting with HIF prolyl hydroxylase 2 (HPH-2), ING4 has been described to repress some HIF1α activities under hypoxic conditions.¹² Here, we show that ING1b accumulates in ADSCs following DNA damage in hypoxia. According to the opposing roles of ING1b and HIF1α, we hypothesized that ING1b could interfere with HIF1α and participate in the conservation of the genomic integrity of MSCs. Mechanistically, we found that ING1b interacted with HIF1α and promoted its proteasomal degradation in hypoxia. SUMOylation of ING1b played a role since the unSUMOylated form of ING1b was unable to trigger HIF1α degradation. The E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4) participated in HIF1α degradation and ING1b accumulation following a genotoxic stress in 1% O₂. ING1b, subsequently, took part in decreasing PIAS4 levels after DNA damage. Finally, we report that ING1b by decreasing HIF1α level modulated ADSC differentiation potential. These data indicate that ING1b, according to its SUMOylation status, regulates the hypoxic response by contributing to the HIF1α degradation, and therefore may impede HIF1α-related effects on the maintenance of ADSCs stem cell character.     

Human meniscus cells express hypoxia inducible factor-1α and increased SOX9 in response to low oxygen tension in cell aggregate culture

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)α(I) in response to 5% oxygen, and this hypoxia-induced expression of P4Hα(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1α inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1α gene and downstream target genes (namely, those encoding P4Hα(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation.     

The increased expression of DEC1 gene is related to HIF-1α protein in gastric cancer cell lines

Overexpression of differentiated embryo chondrocyte 1 (DEC1) has been reported to contribute to the cellular differentiation, proliferation, and apoptosis of various cancers. Our previous studies have shown that DEC1 was highly expressed in gastric cancer (GCa) tissues. However, there is no report about the expression of DEC1 in GCa cell lines until now. In this study, We evaluated the mRNA and protein expression of DEC1 and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions in six GCa cell lines: BGC-823, MGC80-3, MKN1, AGS, FU97 and SGC-7901. An HIF-1α protein inhibitor was used to analyze the association of DEC1 and HIF-1α expression. Under normoxia, the mRNA expression of both HIF-1α and DEC1 was moderate, whereas the protein expression of DEC1 was higher than that of HIF-1α. Hypoxia induced the mRNA expression of DEC1 and the protein expression of HIF-1α and DEC1 in a time-dependent manner but had no effect on the mRNA expression of HIF-1α. Furthermore, inhibition of HIF-1α protein expression resulted in a significant decrease in both the mRNA and protein expression of DEC1. Taken together, DEC1 expression is correlated with HIF-1α protein in GCa cell line, blockage of HIF-1α protein led to reduced DEC1 expression. The efficacy of inhibiting HIF-1α and DEC1 expression should be tested in clinical trials as possible treatment for GCa.     

BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response

Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.     

Immunohistochemical assessment of intrinsic and extrinsic markers of hypoxia in reproductive tissue: differential expression of HIF1α and HIF2α in rat oviduct and endometrium

Hypoxia is thought to be critical in regulating physiological processes within the female reproductive system, including ovulation, composition of the fluid in the oviductal/uterine lumens and ovarian follicle development. This study examined the localisation of exogenous (pimonidazole) and endogenous [hypoxia inducible factor 1α and 2α (HIF1α, -2α), glucose transporter type 1 (GLUT1) and carbonic anhydrase 9 (CAIX)] hypoxia-related antigens within the oviduct and uterus of the rat reproductive tract. The extent to which each endogenous antigen co-compartmentalised with pimonidazole was also assessed. Female Wistar Furth rats (n = 10) were injected intraperitoneally with pimonidazole (60 mg/kg) 1 h prior to death. Reproductive tissues were removed immediately following death and fixed in 4% paraformaldehyde before being embedded in paraffin. Serial sections were cut (6–7 μm thick) and antigens of interest identified using standard immunohistochemical procedures. The mucosal epithelia of the ampulla, isthmus and uterus were immunopositive for pimonidazole in most sections. Co-compartmentalisation of pimonidazole with HIF1α was only expressed in the mucosa of the uterus whilst co-compartmentalisation with HIF2α was observed in the mucosa of the ampulla, isthmus and uterus. Both GLUT1 and CAIX were co-compartmentalised with pimonidazole in mucosa of the isthmus and uterus. This study confirms that mucosal regions of the rat oviduct and uterus frequently experience severe hypoxia and there are compartment specific variations in expression of endogenous hypoxia-related antigens, including the HIF isoforms. The latter observation may relate to target gene specificity of HIF isoforms or perhaps HIF2α’s responsiveness to non-hypoxic stimuli such as hypoglycaemia independently of HIF1α.     

Blocking HIF1α activity eliminates hematological cancer stem cells

Selective targeting of cancer stem cells (CSCs) has the potential to prevent cancer relapse. Wang et?al. (2011) report that hypoxia-inducible factor 1α (HIF1α) represses Notch signaling to maintain CSC subsets from lymphoma, and that blocking HIF1α activity eliminates lymphoma and human acute myeloid leukemia (AML) CSCs.     

Metabolic depression: a response of cancer cells to hypoxia?

Hypoxic tumours have the worst prognosis because they are the most aggressive and the most likely to metastasize. This may be because these aggressive cancers have a hypoxic core which generates signals that activate angiogenesis which enables the supply of nutrients and oxygen to a rapidly growing outer oxidative shell. The hypoxic core is a crucial element of this hypothesis, as is the fact that the cells in the hypoxic core are inherently adapted to survive hypoxia. We reasoned therefore that cancer cells exposed to hypoxia/anoxia should show the hallmarks of adaptation to hypoxia/anoxia, i.e. a down-regulation of protein synthesis and a reverse Pasteur effect. We tested this hypothesis in transformed (MCF-7) and normal (HME) human mammary epithelial cells, by exposing both cell types to a range of oxygen concentrations, including anoxia. We find that indeed protein synthesis is down-regulated in the MCF-7, but not in the HME cells in response to anoxia. The data on glycolysis are not as clear-cut, but in the light of similar previous measurements on hypoxia-tolerant animals, is still consistent with the hypothesis.     

Hypoxia-inducible factor-1α protein negatively regulates load-induced bone formation

Mechanical loads induce profound anabolic effects in the skeleton, but the molecular mechanisms that transduce such signals are still poorly understood. In this study, we demonstrate that the hypoxia-inducible factor-1α (Hif-1α) is acutely up-regulated in response to exogenous mechanical stimuli secondary to prostanoid signaling and Akt/mTOR (mammalian target of rapamycin) activation. In this context, Hif-1α associates with β-catenin to inhibit Wnt target genes associated with bone anabolic activity. Mice lacking Hif-1α in osteoblasts and osteocytes form more bone when subjected to tibia loading as a result of increased osteoblast activity. Taken together, these studies indicate that Hif-1α serves as a negative regulator of skeletal mechanotransduction to suppress load-induced bone formation by altering the sensitivity of osteoblasts and osteocytes to mechanical signals.     

Integrin β4 regulates SPARC protein to promote invasion

The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of β4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates β4-mediated invasion. Expression of β4 in β4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous β4, miR-29a expression is low and β4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that β4 can regulate SPARC expression and that SPARC is an effector of β4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.     

FERM domain-containing protein FRMD5 regulates cell motility via binding to integrin β5 subunit and ROCK1

FRMD5 is a novel FERM domain-containing protein depicted in tumor progression. However, the mechanisms underlying FRMD5 inhibition of cell migration is largely unknown. Here, we show that FRMD5 regulates cell migration by interacting with integrin β5 cytoplasmic tail and ROCK1 in human lung cancer cells. FRMD5 promotes cell–matrix adhesion and cell spreading on vitronectin, and thus inhibits cell migration. Furthermore, FRMD5 interacts with ROCK1 and inhibits its activation that leads to the inhibition of myosin light chain phosphorylation and the actin stress fiber formation. Taken together, these findings demonstrate that the putative tumor suppressive protein FRMD5 regulates tumor cell motility via a dual pathway involving FRMD5 binding to integrin β5 tail and to ROCK1.