상단 PDF The expression of Heat Shock Protein 70 in the placental tissue with preeclampsia

연구대상 및 방법 : 아주대학교 병원 산부인과에서 제왕절개술로 분만한 산모들 중 태아발육부전을 동반하지 않은 경증 및 중증 자간전증 산모 각각 12 명과 자간전증을 동반하지 않은 정상 산모 12 명을 대상으로 하여 분만 후 태반 조직을 채취하였다. 동결조직에 1:50 Heat shock protein 70 일차 항체를 사용하여 면역조직화학적 염색을 하였고 합포체영양막, 세포영양막, 태반기저부의 융모외영약막, 태반기저부 혈관의 내피세포, 탈락막에서 Hsp70 의 발현 정도를 관찰하였으며 강도에 따라 세 환자군을 비교하였다. Western blot 분석을 하여 정상 임신과 전자간증 태반에서의 Hsp70 정량을 비교하였다.

Vector construction The detailed procedure to construct MFG.B7.puro vector, which was used for constructing MFG.Hsp70puro, has been published elsewhere (Kwak et al 1998; Park et al 2000). To establish cell line, 1 mg of MFG.Hsp70puro or the MFGpuro plasmids was introduced into cells by li- pofection (Lipofectamine, GIBCO BRL) in serum-free me- dia. Cells were propagated in DMEM supplemented with 10% FCS, 1% antibiotic/antimycotic solution, 2 mM L - glutamine, and 2 mg/mL of puromycin (all from GIBCO BRL). Twenty-four hours after transfection, media were changed and the cells were maintained in medium con- taining 10% serum and 2 mg/mL of puromycin. Control cells were transfected with MFGpuro alone. hsp70-trans- fected cells were frequently tested for the expression of Hsp70 by Western blot analysis and found to express high levels of inducible Hsp70 protein.

Differential protein expression in Wild type and Hsp70 Tg mice. Proteomic studies to data, other than in biomedicine, have focused mainly on changes in genome expression that are triggered by environmental factors. As shown in 2-D gel, we could clearly separate the up-regulated and down-regulated proteins, ranging from low to high molecular weights. Most proteins were separated in the isoelectric point range of 4-7. Proteins extracted from untreated control were used as the control for comparative analysis. About 900 protein spots were detected in Hsp70 Tg and WT mice with computer-aided image analysis of 2-D gel. 114 protein spots were differently expressed when stained with silver (Figure 4).

Hsp70 function. Our studies revealed that hFAF1 did not bind to the peptide binding domain, substrate-binding sites of Hsp70, but rather bound to N terminus of the ATPase domain of Hsp70, as co-chaperones Bag-1 and Hip bind to the ATPase domain. Also the expression level of hFAF1 was not varied in various stresses, indicating that hFAF1 is not a substrate of Hsp70. We therefore examined whether hFAF1 is involved in the regulation of Hsp70 activity. Transient overexpression of hFAF1 inhibited the chaperone activity of Hsp70, whereas transient expression of the N-terminally deleted hFAF1 mu- tant showed the opposite effect, indicating that hFAF1 may act as a co-chaperone and inhibit the chaperone activity of Hsp70. Recently, co-chaperones of Hsp70 such as Hsp40, Hip, Hop, Bag-1, CHIP, Chap1/PLIC-2, and Chap2/Bat3/Scythe have been identified. Hip and Hop facilitates refolding of unfolded proteins and Bag-1, CHIP, and Chap2/Bat3/Scythe attenuated the chaperone activity of Hsp70 (9, 14, 16, 17–20, 30). Because the latter proteins contain ubiquitin-like do- mains, which bind to proteasomes, they are thought to be involved in the degradation of Hsp70 substrates in protea- somes. Bag-1 contains a ubiquitin-like domain which binds to proteasome; CHIP has U box domain that has ubiquitin li- gase activity, and Chap2/Bat3/Scythe also contains a ubiq- uitin-like domain. The whole sequence of FAF1 is 78 –95%

Hee-Jung Kim‡, Eun Joo Song‡, and Kong-Joo Lee§ From the Center for Cell Signaling Research, Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea Heat shock (HS) induces a wide variety of biological processes, including inhibition of protein synthesis, ele- vated expression of heat shock proteins, induction of thermotolerance, and apoptotic cell death in a dose-de- pendent manner. We compared phosphorylated proteins in heat-shocked and thermotolerant cells using proteome analysis. After HS treatment of control RIF-1 and their thermotolerant derivatives, TR-RIF-1 cells, cellular pro- teins were separated by two-dimensional gel electro- phoresis and the phosphorylated proteins were detected with the anti-phosphotyrosine antibodies. We found that 93 proteins showed significant changes in phosphoryla- tion between control and thermotolerant cells as a func- tion of recovery time after HS; we identified 81 of these proteins with peptide mass fingerprinting using MALDI- TOF MS after in-gel trypsin digestion. These phosphoryl- ated proteins exhibit various cellular functions, including chaperones, ion channels, signaling molecules, in tran- scription and translation processes, in amino acid biosyn- thesis, oxidoreduction, energy metabolism, and cell motil- ity or structure, suggesting that HS turns on the various signaling pathways by activating protein-tyrosine ki- nases (PTKs). Of these, 20 proteins were previously iden- tified phosphorylated proteins and 64 were newly identi- fied. These proteins can be grouped into three families: 1) proteins highly phosphorylated in TR-RIF-1 cells at basal level and phosphorylated more significantly by HS in RIF-1 than TR-RIF-1; 2) proteins highly phosphorylated in control RIF-1 cells at basal level and phosphorylated more easily by HS in TR-RIF-1 than in RIF-1 cells; and 3) proteins with a similar basal phosphorylation level in both RIF-1 and TR-RIF-1 cells and responding to HS sim- ilarly in both cells. Most of the phosphorylated proteins are presumably involved in HS signaling in different ways, with the first and second families of proteins influ- encing thermotolerance. The possible tyrosine phospho- rylation sites, the possible PTKs phosphorylating these proteins, and the proteins binding to these phosphoryl- ated sites were predicted by the Netphos, ScanProsite, and Scansite programs. These results suggest that HS can activate various PTKs and HS responses can be regulated by phosphorylations of proteins having various functions.

Action of AngII are mediated by angiotensin type 1 (AT1) and angiotensin type 2 (AT2) receptors, which are G-protein coupled seven transmembrane glycoproteins. AT1 receptor is present in many tissues and organs, including the heart, blood vessels, kidney, and adipocytes, whereas the AT2 receptor is expressed mainly in the fetus and has low levels of expression after birth. AT1 receptor is responsible for most of the pathophysiological actions of AngII. 4 Among other pathological factors, AngII is an important humoral factor responsible for cardiac hypertrophy, and is associated with the induction of immediate-early genes such as c-fos, c-jun and Egr- 1 which is followed by activation of late genes such as β-myosin heavy chain, α- skeletal and α-smooth muscle actin and atrial natriuretic factor (ANF). 2 Ang II- induced cellular hypertrophy is mainly mediated via stimulation of AT1 receptor. So, pharmacological agents that antagonize the RAS at the level of ACE or the AT1 receptor have come into prominence in the treatment of hypertension, cardiac hypertrophy, heart failure and cardiac remodeling. Practically, administration of either an ACE inhibitor or an AT1 receptor antagonist not only significantly induces the regression, but also prevents the development of cardiac hypertrophy in experimental animal models. Previous studies have shown that AngII also induces myocyte apoptosis.

DISCUSSION Chronic infections may trigger, sustain, or exacerbate autoimmune disease through epitope spreading via the antigen presentation of epitopes released within injured tissue [13]. Identification of the target antigenic peptide of an infecting pathogen responsible for epitope spreading may facilitate the development of a vaccination strategy for the suppression of autoimmune disease [8]. Hagiwara et al. [7] recently suggested that sublingual immunization with GroEL significantly suppresses atherosclerosis induced by naturally-occurring bacteria, inflammatory cytokines, and oxLDL levels accelerated by P. gingivalis infection. In the present study, we found that nasal immunization with Pep14, a peptide derived from PgHSP60, could attenuate Pep14-induced atherosclerotic plaque formation in an ApoE KO mouse model and confer atheroprotection equivalent to that achieved via whole HSP60 immunization [7]. We propose that nasal immunization with Pep14 would be preferable to using a whole

transfection or PGF 2α treatment leads to an increase in OxLDL-induced Hsp70 protein expression. Also, siRNA inhibition of PPARγ reversed the troglita- zone-mediated inhibition of Hsp70 protein expre- ssion. Collectively, these results suggest that OxLDL and TZDs may inhibit translation by PPARγ- dependent as well as PPARγ-independent mecha- nisms in monocytes/macrophages. Recent studies suggest that PPARγ can suppress the function of other transcription factors, such as the nuclear receptor co-repressor (NCoR)-histone deace- tylase-3 (HDAC3) complexes, by direct interaction (Pascual et al., 2005). Therefore, it is possible that PPARγ activation leads to the suppression of tran- slation by binding to translation machinery proteins under oxidative stress conditions as well. It remains to be elucidated whether PPARγ regulates trans- lation through a direct interaction with translation factors, and, if so, which factors. Thus, molecular mechanisms underlying anti-inflammatory properties of PPARγ may include not only transrepression of NF-κB-mediated transcription but also suppression of de novo synthesis of stress-induced pro-inflam- matory proteins.

4. Establishment and characterization of P. gingivalis heat shock protein-specif- ic T cell lines PP.. ggiinnggiivvaalliiss -specific T cell lines from peripheral blood were established according to the method previously described 20-23 . Peripheral blood mononu- clear cells were also isolated by gradient cell separa- tion technique using Ficoll-Paque medium (Pharmacia, Upsala, Sweden). Using 12-well tissue culture plates (Costar, Corning, Corning, NY), mononuclear cells (1 x 10 6 cells/well) isolated from peripheral blood were stimulated with P. gingivalis (1 x 10 8 cells/ml) together with antigen presenting cells (APC, 5 x 10 6 cells/well) after treatment with mitomycin C (25 mg/ml). Peripheral blood lympho- cytes were additionally drawn from the patients, T cells were removed by nylon wool, and non-T cell fraction was used as APCs. After 2 weeks of incuba- tion, T cells were allowed to rest for 1 week. After the resting period, fresh mitomycin-treated non-T cells and P. gingivalis were added again to induce T

In the present study, we investigated the cross-species effects of Hsp22. We report that the DmHsp22 is functionally active in human cells. Indeed, the expression of DmHsp22 extends the life span of normal human fibroblasts and increased the malig- nant properties of human cancer cells. Moreover, DmHsp22- expressing cancer cells formed aggressive tumors and acquired a drug-resistant phenotype. We further report that DmHsp22 interacts with and inactivate wild type tumor suppressor p53 in human cancer cells, suggesting that this interaction may repre- sent one of the mechanisms of its proproliferative function.

DISCUSSION An important protein in cell biology, HSP was well preserved during evolution. Its production is initiated by various stressful stimuli including heat, ultraviolet irradiation, heavy metals, infection, inflammation, hypoxia, tissue damage, and tumors. HSP27 controls dynamic changes in actin and apoptosis. HSP27 reacts to the cytochrome c/Apaf-1/dATP complex in the procaspase 9 pathway and partially inhibits connectivity between Fas, Table 3. Averages of HSP27 andc-FLIP expression according to Gleason score and pathologic stage (n=83)

주제어 : 오리, 열 스트레스, 대사에너지, 융모, 미생물, 유전자 Abstract : The object of this study was to determine the influence of dietary metabolic energy (ME) on ..... A total of 240 meat ducks Cherry valley ( Anas platyrhynchos ) were assigned into four treatment groups with a randomized block design for 42 days. The four treatments were: ME 2900 kcal/kg, ME 3000 kcal/kg, ME 3100 kcal/kg, and ME 3200 kcal/kg. There was no difference in liver tissue among the treatments. The duodenal villi and crypt depth length decreased by 10.58% in 2900 compared with ME 3000, but there was no difference between 3100 and 3200.

shock response (HSR) is elicited. To better understand the molecular regulation of the HSR, we used 2D-PAGE– based pro- teome analysis to screen for heat shock–induced post-transla- tionally modified cellular proteins. Our analysis revealed that two protein spots typically present on 2D-PAGE gels and containing heterogeneous nuclear ribonucleoprotein K (hnRNP K) with trioxidized Cys 132 disappeared after the heat shock treatment and reappeared during recovery, but the total amount of hnRNP K protein remained unchanged. We next tested whether hnRNP K plays a role in HSR by regulating HSF1 and found that hnRNP K inhibits HSF1 activity, resulting in reduced expression of hsp70 and hsp27 mRNAs. hnRNP K also reduced binding affinity of HSF1 to the heat shock element by directly interacting with HSF1 but did not affect HSF1 phosphoryla- tion– dependent activation or nuclear localization. hnRNP K lost its ability to induce these effects when its Cys 132 was substi- tuted with Ser, Asp, or Glu. These findings suggest that hnRNP K inhibits transcriptional activity of HSF1 by inhibiting its bind- ing to heat shock element and that the oxidation status of Cys 132 in hnRNP K is critical for this inhibition.

Our labs showed that MMP-9, one of several genes re- gulated by NF-kB, was reduced in cultured Hsp70-over- expressing astrocytes exposed to ischemia-like insults. Con- sistent with the notion that Hsp70 may regulate inflammatory protein expression at the transcriptional level, MMP-9 mRNA was also lower in Hsp70-transfected cells. However, Hsp70 expressed in astrocytes seems to not only decrease expression of MMP-9 at both the transcriptional and translational level, it also decreased MMP-2 [54]. Interestingly, MMP-9 expression is regulated by NF-kB, whereas MMP-2 is not, suggesting that Hsp70 may interfere with transcriptional responses in systems other than NF-kB. In fact, studies in alveolar macrophages suggest that heat stress-induced Hsp70 can inhibit STAT1 [55], and STAT1 has been linked to MMP-2 expression [56].

4 degradation in a Smurf2 ubiquitin E3 ligase-dependent manner, thus preventing Smad2/3 activation. 13, 15 TGF- β receptor I and II specifically interact with Hsp90 and are clients of this cellular chaperone. 13 Recently, the overexpression of Hsps indicates that both a proliferative (hsp70) and a collagen synthesis (hsp47, hsp27) component are present in keloid tissue. 16 Hsp47 has been reported to be upregulated in keloid fibroblasts andcould induce excessive collagen accumulation by enhancing synthesis and secretion of collagen. 17 Also, we demonstrated that Hsp70 is overexpressed in keloid fibroblasts and tissue and this overexpression of Hsp70 may be involved in the pathogenesis of keloids. 18 However, the clinical significance of Hsp90 inhibitors such as 17AAG in disease models with aberrant TGF-b responses such as keloid and hypertrophic scar remains to be determined.

Dendritic cells play an important role in determining whether naïve T cells mature into either Th1 or Th2 cells. We de- termined whether heat-shock protein X (HspX) purified from Mycobacterium tuberculosis regulates the Th1/Th2 immune response in an ovalbumin (OVA)-induced murine model of asthma. HspX increased interferon-gamma, IL-17A, -12 and transforming growth factor (TGF)- production and T-bet gene expression but reduced IL-13 production and GATA-3 gene expression. HspX also inhibited asthmatic reactions as demon- strated by an increase in the number of eosinophils in bron- choalveolar lavage fluid, inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyper-respon- siveness. Furthermore, HspX enhanced OVA-induced decrease of regulatory T cells in the mediastinal lymph nodes. This study provides evidence that HspX plays critical roles in the amelioration of asthmatic inflammation in mice. These find- ings provide new insights into the immunotherapeutic role of HspX with respect to its effects on a murine model of asthma.

Ⅳ. 고 찰 HSP70의 패혈증에서의 역할 및 이의 임상적 적용 가능성 에 대해서는 아직 확실하게 정립되고 있지 못한 상태이다. 세계적으로도 패혈증에 대한 HSP70의 예방적 효과에 대한 연구만 시행되어 왔으며 실제 임상에 적용할 수 있는 방법 은 아직 개발되지 못하고 있다. Heat shock, GGA 등의 전 처치를 통해서는 생존 향상 및 예후의 호전을 가져올 수 있 으나 오히려 패혈증 유발 후 heat shock treatment를 가했을 경우에는 HSP70의 감소 및 예후의 악화를 보고하고 있어 이를 heat shock paradox라고 보고하고 있다 41 . 다만 패혈증 유발 후 GLN의 투여 등 간접적으로 HSP70이 과도 발현된 치료 효과에 대한 결과는 보고되고 있다 42 . 또한 GLN의 투 여는 iNOS mRNA, nuclear factor (NF)-κB의 발현 및 활성의

Conclusions The observations presented here establish correlations between Hypo and reduced rod formation and between Hsp70 and reduced rod formation after brain ischemia, but they do not prove that reduced rod formation is a major factor mediating either beneficial effect. However, our observations that rod formation is reduced under the conditions of neuroprotection indicate that their presence is likely to be more harmful than beneficial in brain ischemia. Further, mechanisms of injury to neuronal processes (dendrites and axons) are poorly understood, and cofilin rod formation is becoming increasingly recognized as both a marker and participant in their pathology. Ongoing studies using genetic and pharmacological means of suppressing cofilin rod formation may be able to resolve this contribution of suppressed rod formation to preservation of neuronal processes by Hypo and Hsp70.

In the present study, all affected family members had similar clinical phenotypes and disease pro- gression patterns. The first presenting motor symp- tom was paresis of extensor muscles of the great toe and later of extensor muscles of the other toes and of the feet. The disease initially paralyses distal muscles of the lower extremities, and pro- gresses to cause muscle weakness and atrophy in the hands and proximal leg muscles. Sensory abnormalities and pyramidal tract signs were absent in all cases, as was pes cavus. In all family members, the disease pursued a slowly progressive course that caused disability of proximal thigh muscles after 20 years of evolution.

2.2. Mechanism action of topoisomerase inhibitor (Topi) Topoisomerase inhibitor (Topi)는 Top1 및 Top2를 표적으로 하여 DNA의 전사 및 복제 과정을 방해하는 억제제이다 [7,12]. Topi는 크게 4가지 기전을 통해 topoisomerase를 억제 하여 항암 효과를 나타낸다. 첫 번째는 topoisomerase의 활성 부위에 inhibitor가 결합하여 DNA 기질이 결합하는 것을 방 해하는 기질 경쟁 억제 식이지만 아직 주목할 만한 예시가 없 다. 두 번째는 ‘Topoisomerase poisons’을 형성하는 것으로 가 장 활발하게 연구가 되었다. Topi의 결합에 따라 Ternary protein-DNA-drug complex 로 구성되며 DNA의 재결합을 방 지하고 효소를 절단 부위에 고정해 효소의 전환을 막아 DNA 복제 억제, 이중 가닥 절단 및 후속 세포 사멸 등의 독 성을 주게 된다. 세 번째는 촉매 억제제를 통해 효소 활성을 막는 것이다. 이는 Top1B와 Top2A 대상으로 한 촉매 억제제 로써 임상 단계에서 큰 주목을 받지 못했다. 하지만 암세포 에서 다량의 topoisomerase를 억제할 경우 촉매 억제제는 강 력한 항암제가 될 수 있을 것으로 보인다. 마지막으로 ATP Fig. 1. Mechanism of topoisomerase, HSP90, mTOR and tyrosine