Chaperoning stem cells: a role for heat shock proteins in the modulation of stem cell self‐renewal and differentiation?

Self‐renewal and differentiation of stem cells are tightly regulated processes subject to intrinsic and extrinsic signals. Molecular chaperones and co‐chaperones, especially heat shock proteins (Hsp), are ubiquitous molecules involved in the modulation of protein conformational and complexation states. The function of Hsp, which are typically associated with stress response and tolerance, is well characterized in differentiated cells, while their role in stem cells remains unclear. It appears that embryonic stem cells exhibit increased stress tolerance and concomitant high levels of chaperone expression. This review critically evaluates stem cell research from a molecular chaperone perspective. Furthermore, we propose a model of chaperone‐modulated self‐renewal in mouse embryonic stem cells.     

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated β-galactosidase (SA-β-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.     

The altered expression of glucose-regulated proteins 78 in different phase of streptozotocin-affected pancreatic beta-cells

Endoplasmic reticulum (ER) stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cells and contributes to the development of type 1 diabetes. The chaperone molecule, glucose-regulated proteins 78 (Grp78), is required to maintain ER function during toxic insults. In this study, we investigated the changes of Grp78 expression in different phases of streptozotocin (STZ)-affected beta-cells to explore the relationship between Grp78 and the response of beta-cells to ER stress. An insulinoma cell line (NIT-1) treated with STZ for different time periods and STZ-induced diabetic Balb/C mice at different time points were used as the model system. The level of Grp78 and C/EBP homologous protein (CHOP) mRNA were detected by real-time polymerase chain reaction and their protein by immunoblot. Apoptosis and necrosis was measured by flow cytometry. In addition, the changes of Grp78 protein in STZ-treated nondiabetic mice were also detected by immunoblot. Grp78 expression significantly increased in the early phase but decreased in the later phase of affected beta-cells, while CHOP was induced and apoptosis occurred along with the decrease of Grp78. Interestingly, the Grp78 protein of STZ-treated nondiabetic mice increased stably compared with that of the control. From the results, we can conclude that Grp78 may contribute to the response of beta-cells to ER stress, and more attention should be paid to Grp78 in the improvement of diabetes.     

The action of Pdcd4 may be cell type specific: evidence that reduction of dUTPase levels might contribute to its tumor suppressor activity in Bon-1 cells

Pdcd4 (programmed cell death protein 4) was identified as a gene up-regulated during apoptosis and, additionally, seems to have a function as a tumor suppressor. However, there are conflicting data concerning its role in programmed cell death and most results for its action as an inhibitor for neoplastic transformation are derived from experiments with epidermal cells. Therefore, we were interested to investigate if the action of Pdcd4 might be cell type specific. For that purpose we examined the expression of Pdcd4 and several other proteins in various tumor cell lines. We could not find any correlation of Pdcd4 levels and expression of proteins associated with cell cycle and/or apoptosis in different cell lines. Furthermore, we stably transfected two cell lines (Bon-1 and HCT116) to over-express Pdcd4 and analyzed protein expression. Although we found several regulated proteins none of these proteins were affected in both cell lines in the same manner. For instance, dUTPase expression was reduced in Bon-1 cells but not changed in HCT116 cells. This regulation might be important for the sensitivity of cells to anti-cancer drugs like inhibitors of thymidilate synthase. Therefore, we conclude that the function of Pdcd4 might be cell type specific. A role for Pdcd4 in apoptosis or as a tumor suppressor might be limited to certain cell types.     

Gadd45 in stress signaling

Gadd45 genes have been implicated in stress signaling in response to physiological or environmental stressors, which results in cell cycle arrest, DNA repair, cell survival and senescence, or apoptosis. Evidence accumulated implies that Gadd45 proteins function as stress sensors is mediated by a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and the response of cells to stress. These include PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. What deterministic factors dictate whether Gadd45 and partner proteins function in either cell survival or apoptosis remains to be determined. An attractive working model to consider is that the extent of cellular/DNA damage, in a given cell type, dictates the association of different Gadd45 proteins with particular partner proteins, which determines the outcome.     

Death by chaperone: HSP90, HSP70 or both?

HSP70 family members are highly conserved proteins that function as molecular chaperones. Their principle role is to aid protein folding and promote the correct cellular localisations of their respective substrates. The function of HSP70 isoforms can be exhibited independently or with the HSP90 chaperone system in which HSP70 is important for substrate recruitment. In addition to their chaperone role, HSP70 isoforms promote cell survival by inhibiting apoptosis at multiple points within both the intrinsic and extrinsic cell death pathways. Consistent with this cytoprotective function, increased expression of HSP70 isoforms is commonly associated with the malignant phenotype. We recently reported that dual silencing of the major constitutive (HSC70) and inducible (HSP72) isoforms of HSP70 in cancer cells could phenocopy the effects of a pharmacologic HSP90 inhibitor to induce proteasome-dependent degradation of HSP90 client proteins CRAF, CDK4 and ERBB2. This was accompanied by a G1 cell cycle arrest and extensive apoptosis which was not seen in non-tumorigenic human cell lines. Here we discuss the possible implications of our research for the development of HSP70 family modulators which offer not only the possibility of inhibiting HSP70 activity but also the simultaneous inhibition of HSP90, resulting in extensive tumour-specific apoptosis.     

The role of stress proteins in prostate cancer

The development of therapeutic resistance, after hormone or chemotherapy for example, is the underlying basis for most cancer deaths. Exposure to anticancer therapies induces expression of many stress related proteins, including small heat shock proteins (HSPs). HSPs interact with various client proteins to assist in their folding and enhance the cellular recovery from stress, thus restoring protein homeostasis and promoting cell survival. The vents of cell stress and cell death are linked, as the induction of molecular chaperones appears to function at key regulatory points in the control of apoptosis. On the basis of these observations and on the role of molecular chaperones in the regulation of steroid receptors, kinases, caspases, and other protein remodelling events involved in chromosome replication and changes in cell structure, it is not surprising that molecular chaperones have been implicated in the control of cell growth and in resistance to various anticancer treatments that induce apoptosis. Recently, several molecular chaperones such as Clusterin and HSP27 have been reported to be involved in development and progression of hormone-refractory prostate cancer. In this review, we address some of the molecular and cellular events initiated by treatment induced stress, and discuss the potential role of chaperone proteins as targets for prostate cancer treatment.     

The small heat shock protein alpha B-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor alpha family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein alpha B-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type alpha B-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of alpha B-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type alpha B-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation alpha B-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that alpha B-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of alpha B-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer.     

The chaperone function of hsp70 is required for protection against stress-induced apoptosis

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.     

Heat induced stress proteins and the concept of molecular chaperones

Heat stress proteins can be assigned to eleven protein families conserved among bacteria, plants and animals. Most of them aid other proteins to maintain or regain their native conformation by stabilizing partially unfolded states. Hence, they are called molecular chaperones. Experimental data indicate that many of them form heterooligomeric complexes, so-called chaperone machines, interacting with each other to generate a network for maturation, assembly and intracellular targeting of proteins. In this review we summarize the essential information on the structure and function of chaperone and chaperone complexes. In addition we present a compilation ofin vivo andin vivo test systems used in the preceding ten years of chaperone research.     

NOX家族蛋白的研究进展

NADPH氧化酶催化亚基gp91phox（NOX2）及其同源物NOX1、NOX3、NOX4、NOX5、DUOX1和DUOX2统称为NOX家族,它们作为NADPH酶的核心亚基,是该酶发挥作用的关键。NOX家族几乎存在于所有的细胞,吞噬细胞中NADPH氧化酶生成的ROS主要起细胞防御功能,与此不同的是非吞噬细胞中NADPH氧化酶产生的ROS作为信号分子,参与机体内信号转导途径,调节细胞分化、增殖、衰老和凋亡等活动;当NOX家族蛋白异常表达,ROS水平急剧增加时,则能诱导机体内多种疾病的发生。     

In vitro evidence of Hsc70 functioning as a molecular chaperone during cold stress

Hsp70 molecular chaperones have been shown to play an important role in helping cells to cope with adverse environments, especially in response to high temperatures. The molecular chaperone function of Hsc70 at low temperature was investigated. A cold-inducible spinach cytosolic Hsc70 was subcloned into a protein expression vector and the recombinant protein was expressed in bacterial cells. Recombinant Hsc70 bound a permanently unfolded substrate: alpha-carboxymethylated lactalbumin (CMLA) in the presence of 3 mM ATP and MgCl(2) at low temperature (4 and -4 degrees C). Radiolabeling with (35)S-Met and (35)S-Cys and immunoprecipitation with cytosolic Hsc70 monoclonal antibodies showed that there were several proteins co-immunoprecipitated at low temperature (4 and -4 degrees C) but not at room temperature. Enhanced co-purification of sHsp17.7 with Hsc70 at low temperature was observed and suggests that co-chaperone interactions can contribute to molecular chaperone function during cold stress. These results suggest that the molecular chaperone Hsc70 may have a functional role in plants during low temperature stress.     

热激蛋白70研究进展

热激蛋白70（HSP70）是广泛存在且高度保守的蛋白,作为伴侣分子能够促进蛋白折叠;HSP70可以通过阻止细胞色素c从线粒体释放,与凋亡诱导因子结合使其不能入核,或者抑制JNK激酶活性调节细胞凋亡;HSP70可以调节细胞周期进程,促进细胞生长,阻止细胞衰老;免疫功能研究表明HSP70是有效的免疫佐剂,可激发抗原特异性的CTL反应,同时细胞外HSP70和膜结合HSP70可激发非特异性免疫反应。     

Induction of cellular senescence by insulin-like growth factor binding protein-5 through a p53-dependent mechanism

The insulin-like growth factor (IGF) signaling pathway plays a crucial role in the regulation of cell growth, differentiation, apoptosis, and aging. IGF-binding proteins (IGFBPs) are important members of the IGF axis. IGFBP-5 is up-regulated during cellular senescence in human dermal fibroblasts and endothelial cells, but the function of IGFBP-5 in cellular senescence is unknown. Here we show that IGFBP-5 plays important roles in the regulation of cellular senescence. Knockdown of IGFBP-5 in old human umbilical endothelial cells (HUVECs) with IGFBP-5 micro-RNA lentivirus caused partial reduction of a variety of senescent phenotypes, such as changes in cell morphology, increases in cell proliferation, and decreases in senescence-associated beta-galactosidase (SA-beta-gal) staining. In addition, treatment with IGFBP-5 protein or up-regulation of IGFBP-5 in young cells accelerates cellular senescence, as confirmed by cell proliferation and SA-beta-gal staining. Premature senescence induced by IGFBP-5 up-regulation in young cells was rescued by knockdown of p53, but not by knockdown of p16. Furthermore, atherosclerotic arteries exhibited strong IGFBP-5-positive staining along intimal plaques. These results suggest that IGFBP-5 plays a role in the regulation of cellular senescence via a p53-dependent pathway and in aging-associated vascular diseases.     

PIAS蛋白家族的研究进展

PIAS(protein inhibitor of activated STAT)蛋白家族是一种能够激活STAT转录活性的抑制蛋白,共包括4个成员,可与多种蛋白发生相互作用,从而影响靶蛋白的活性和功能,其主要与STAT、Wnt、TGF-β、NF-κB等通路的转录因子或转录辅因子相互作用以调控下游基因的转录活性。在细胞周期中,PIAS蛋白是细胞衰老和细胞凋亡的调节子,可促进细胞的扩散和衰老。在肿瘤发生中,PIAS蛋白的过表达能抑制癌细胞的增殖并诱导其凋亡。除此之外,在生殖系统和神经系统中,PIAS家族蛋白也能通过与相关的转录因子或激素受体相互作用影响其发生发展的过程。     

Identification of alpha-tubulin as an hsp105alpha-binding protein by the yeast two-hybrid system

Hsp105alpha is a mammalian stress protein that belongs to the HSP105/110 family. Hsp105alpha prevents stress-induced apoptosis in neuronal cells and binds to Hsp70/Hsc70 and suppresses the Hsp70 chaperone activity in vitro. In this study, to further elucidate the function of Hsp105alpha, we searched for Hsp105alpha-binding proteins by screening a mouse FM3A cell cDNA library with full-length Hsp105alpha using the yeast two-hybrid system and obtained alpha-tubulin as an Hsp105alpha-binding protein. Hsp105alpha bound directly to alpha-tubulin both in vitro and in vivo. Indirect immunofluorescence analysis with anti-Hsp105 and anti-alpha-tubulin antibodies indicated that Hsp105alpha was colocalized with microtubules. Furthermore, the disorganization of microtubules induced by heat shock was prevented in Hsp105alpha-overexpressing COS-7 cells. These findings suggested that Hsp105alpha associates with alpha-tubulin and microtubules in cells and plays a role in protection of microtubules under conditions of stress.     

Bcl-2 promotes premature senescence induced by oncogenic Ras

The expression of the apoptosis inhibitory protein, Bcl-2, is increased in naturally senescing human fibroblasts and upon induction of their senescence-like growth arrest by oxidative stress, implying its role in maintaining their extended viability. Oncogenic Ras(V12) protein induces signaling cascades that result in the premature senescence of primary fibroblast cells, which are insensitive to oncogene-dependent apoptosis. Here we show that constitutive expression of Bcl-2 accelerates selected features of the Ras-induced senescence program in primary human fibroblasts. Yet, Bcl-2 also inhibits fibroblast apoptosis induced by exogenous H(2)O(2), while both signals induce an increased endogenous Bcl-2 expression in these cells. Together, these data suggest a context-dependent phenotypic function of Bcl-2 in the regulation of overlapping cell fate specification programs, with potential implications for both physiology and multistep tumorigenesis.