Phosphorylation of the small heat shock-related protein, HSP20, in vascular smooth muscles is associated with changes in the macromolecular associations of HSP20

Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of a small heat shock-related protein, HSP20. We hypothesized that phosphorylation of HSP20 in vascular smooth muscles is associated with alterations in the macromolecular associations of HSP20. Treatment of bovine carotid artery smooth muscles with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and the adenylate cyclase activator, forskolin, led to increases in the phosphorylation of HSP20 and dissociation of macromolecular aggregates of HSP20. However, 3-isobutyl-1-methylxanthine and forskolin treatment of a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation, human umbilical artery smooth muscle, did not result in increases in the phosphorylation of HSP20 or to dissociation of macromolecular aggregates. HSP20 can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (PKA) in both carotid and umbilical arteries and this phosphorylation of HSP20 is associated with dissociation of macromolecular aggregates of HSP20. Activation of cyclic nucleotide-dependent signaling pathways does not lead to changes in the macromolecular associations of another small heat shock protein, HSP27. Interestingly, the myosin light chains (MLC20) are in similar fractions as the HSP20, and phosphorylation of HSP20 is associated with changes in the macromolecular associations of MLC20. These data suggest that increases in the phosphorylation of HSP20 are associated with changes in the macromolecular associations of HSP20. HSP20 may regulate vasorelaxation through a direct interaction with specific contractile regulatory proteins.     

Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.     

Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins

Cyclosporine A (CSA) is a type 2B phosphatase inhibitor which can induce contraction of renal artery smooth muscle. In this investigation, we examined the phosphorylation events associated with CSA-induced contraction of bovine renal artery smooth muscle. Contractile responses were determined in a muscle bath and the corresponding phosphorylation events were determined with whole cell phosphorylation and two-dimensional gel electrophoresis. CSA-induced contractions were associated with increases in the phosphorylation of the 20 kDa myosin light chains (MLC20) and different isoforms of the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions was associated with increases in the phosphorylation of another small heat shock protein, HSP20, and decreases in the phosphorylation of the MLC20, and some isoforms of HSP27. These data suggest that CSA-induced contraction and relaxation of vascular smooth muscle is associated with increases in the phosphorylation of specific contractile regulatory proteins.     

The small heat shock-related protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotide-dependent relaxation

The small heat shock-related protein 20 (HSP20) is present in four isoforms in bovine carotid artery smooth muscles. Three of the isoforms are phosphorylated and one is not. Increases in the phosphorylation of two isoforms of HSP20 (isoform 3, pI 5.9; and 8, pI 5.7) are associated with cyclic nucleotide-dependent relaxation of bovine carotid artery smooth muscles. Increases in the phosphorylation of another isoform (isoform 4, pI 6.0) are associated with phorbol ester-induced contraction of bovine carotid artery smooth muscles. In this investigation we determined that isoforms 3 and 8 are phosphorylated on Ser16 of the HSP20 molecule during activation of cAMP-dependent signaling pathways. Phosphorylation state-specific antibodies produced against a peptide containing phosphorylated Ser16 recognized isoforms 3 and 8 but not isoform 4. In human vascular tissue, only isoform 3 is present. Incubation of transiently permeabilized strips of bovine carotid artery smooth muscle with synthetic peptides in which Ser16 is phosphorylated, inhibits contractile responses to high extracellular KCl and to serotonin. These data suggest that phosphorylation of HSP20 on Ser16 modulates cAMP-dependent vasorelaxation.     

Stress causes decrease in vascular relaxation linked with altered phosphorylation of heat shock proteins

Cyclic nucleotide-dependent vascular relaxation is associated with increases in the phosphorylation of a small heat shock protein (HSP), HSP20. An increase in phosphorylation of another small HSP, HSP27, is associated with impaired cyclic nucleotide-dependent vascular relaxation. Expression of HSPs is altered by exposure to several types of cellular stress in vitro. To determine if behavioral stress in vivo alters vascular expression and phosphorylation of the small HSPs and cyclic nucleotide-dependent vascular relaxation, borderline hypertensive rats were stressed by restraint and exposure to air-jet stress 2 h/day for 10 days or remained in their home cage. Stress impaired relaxation of aorta to forskolin, which activates adenylyl cyclase, and sodium nitroprusside, which activates guanylyl cyclase. This was associated with an increase in the aortic expression and phosphorylation of HSP27, which was localized to the vascular smooth muscle, but a decrease in the amount of phosphorylated (P)-HSP20. To determine if P-HSP27 inhibits phosphorylation of HSP20, P-HSP27 was added to a reaction mixture containing recombinant HSP20 and the catalytic subunit of cAMP-dependent protein kinase. P-HSP27 inhibited phosphorylation of HSP20 in a concentration-dependent manner. These data demonstrate that P-HSP27 can inhibit phosphorylation of HSP20. The increase in P-HSP27 and decrease in P-HSP20 were associated with reduced cyclic nucleotide-dependent vascular smooth muscle relaxation in response to behavioral stress in vivo, an effect similar to that observed previously in response to cellular stress in vitro.     

Differential expression profiling of the proteomes and their mRNAs in porcine white and red skeletal muscles

Skeletal muscle is an heterogeneous tissue with various biochemical and physical properties of several fiber types. In this study, we carried out the comparative study of protein expression patterns in white and red muscles using two-dimensional gel electrophoresis (2-DE). From more than 500 protein spots detected on each 2-DE gel, we screened five proteins that were differentially expressed between white and red muscles. Using peptide mass fingerprint and tandem mass spectrometry analysis these proteins were identified as myoglobin, two slow-twitch isoforms of myosin light chain and two small heat shock proteins (HSP20 and HSP27). The protein levels of myoglobin, myosin light chain and HSP20 were higher in red muscle, whereas HSP27 was higher in white muscle. In addition, genes of the identified proteins were cloned and their mRNAs were examined. Positive correlations between protein content and their mRNA levels were observed in white and red muscle. These results may provide us with valuable information to understand the different expression profiling between white and red muscle at the protein level.     

Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle.

Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with "focal adhesion complexes" (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli.     

PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.     

热休克蛋白90对血小板源衍生因子诱导的大鼠主动脉平滑肌细胞增殖的影响

目的:研究热休克蛋白90(HSP90)对血小板衍生因子(Platelet derived growth factor,PDGF)诱导的大鼠主动脉平滑肌细胞增殖的影响。方法:采用胶原酶消化法原代培养大鼠胸主动脉平滑肌细胞,应用脂质体细胞转染siRNA的方法抑制HSP90的表达,定量PCR和western blot的方法检测抑制效率。利用PDGF-bb诱导刺激平滑肌细胞增殖,CCK8法检测细胞增殖能力的变化,流式细胞术检测细胞生长周期的改变。结果:平滑肌细胞中转染HSP90的siRNA后,HSP90的mRNA和蛋白水平明显降低,分别为对照组的65.3%和57.6%(P0.05);PDGF-bb刺激明显促进平滑肌细胞生长,而降低HSP90水平显著影响PDGF-bb诱导的细胞增殖(P0.05);流式细胞术检测发现降低HSP90水平引起平滑肌细胞生长停滞,分布在细胞周期G1期的细胞比例明显增多(P0.05)。结论:HSP90通过调控平滑肌细胞的生长周期参与调节细胞增殖过程。     

HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise

Exercise causes heat shock (muscle temperatures of up to 45 degrees C, core temperatures of up to 44 degrees C) and oxidative stress (generation of O2- and H2O2), and exercise training promotes mitochondrial biogenesis (2-3-fold increases in muscle mitochondria). The concentrations of at least 15 possible heat shock or oxidative stress proteins (including one with a molecular weight of 70 kDa) were increased, in skeletal muscle, heart, and liver, by exercise. Soleus, plantaris, and extensor digitorum longus (EDL) muscles exhibited differential protein synthetic responses ([3H]leucine incorporation) to heat shock and oxidative stress in vitro but five proteins (particularly a 70 kDa protein and a 106 kDa protein) were common to both stresses. HSP70 mRNA levels were next analyzed by Northern transfer, using a [32P]-labeled HSP70 cDNA probe. HSP70 mRNA levels were increased, in skeletal and cardiac muscle, by exercise and by both heat shock and oxidative stress. Skeletal muscle HSP70 mRNA levels peaked 30-60 min following exercise, and appeared to decline slowly towards control levels by 6 h postexercise. Two distinct HSP70 mRNA species were observed in cardiac muscle; a 2.3 kb mRNA which returned to control levels within 2-3 h postexercise, and a 3.5 kb mRNA species which remained at elevated concentrations for some 6 h postexercise. The induction of HSP70 appears to be a physiological response to the heat shock and oxidative stress of exercise. Exercise hyperthermia may actually cause oxidative stress since we also found that muscle mitochondria undergo progressive uncoupling and increased O2- generation with increasing temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional expression of NOS 1 in vascular smooth muscle

Substances that increase intracellular calcium concentration ([Ca(2+)](i)), such as serotonin, are known to induce vascular smooth muscle (VSM) contraction. However, increases in [Ca(2+)](i) also activate Ca(2+)/calmodulin-dependent nitric oxide synthases (NOS), which leads to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). One recently identified substrate protein of PKG is the small heat shock protein, HSP20. The purpose of this study was to determine if serotonin activates a Ca(2+)-dependent NOS in VSM. Strips of bovine carotid arterial smooth muscle denuded of endothelium were stimulated with serotonin in the presence and absence of the nonspecific NOS inhibitor N-monomethyl-L-arginine (L-NMMA). Activation of NOS was determined by increases in cGMP and in the phosphorylation of HSP20. Immunohistochemical and Western blotting techniques were performed to identify specific NOS isoforms in bovine carotid arterial smooth muscle preparations. Serotonin stimulation led to significant increases in cGMP and in the phosphorylation of HSP20, which were inhibited by pretreatment with L-NMMA. Antibodies against NOS 1 stained the media of bovine carotid and human renal arteries, whereas antibodies against NOS 3 stained only the endothelium. Additionally, the conversion of radiolabeled L-arginine to L-citrulline NOS activity demonstrated a consistent amount of activity present in the endothelium-denuded smooth muscle preparations that was reduced by 99% with an NOS 1 specific inhibitor. Finally, an NOS 1 specific inhibitor, 7-nitroindazole, augmented contractions induced by high extracellular KCl. This study demonstrates that NOS 1 is present in VSM and may effect physiological contractile responses.     

Exercise-induced elevation of HSP70 is intensity dependent.

Exercise induces expression of the protective heat shock protein, HSP70, in striated muscle. To characterize the relationship between induction of this protein and exercise intensity in muscles exhibiting different recruitment patterns, male Sprague-Dawley rats were assigned to a sedentary control or one of seven exercise groups for which treadmill running speed varied between 15 and 33 m/min (n = 8/group). Twenty-four hours after a single 60-min exercise bout, hearts, red and white portions of the vastus (RV and WV, respectively) muscles, and soleus (Sol) muscles were harvested and analyzed for both relative and absolute HSP70 content. Cardiac HSP70 was significantly elevated only when animals were exercised at 24 m/min and beyond. Similarly, HSP70 was elevated in RV at running speeds above 24 m/min but did not increase in WV until 27 m/min. In contrast, HSP70 content was initially elevated in the Sol but subsequently declined at the highest running speeds. The observed patterns of HSP70 expression in skeletal muscle were in general accordance with known muscle recruitment patterns and suggest that alterations in muscle loading, resulting from changes in exercise intensity, are an important component of exercise-induced increases in HSP70 content.     

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57%+/-4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.     

Heat stress facilitates stretch-induced hypertrophy of cultured muscle cells

Increased mechanical stress induced by stretch is an important growth stimulus in skeletal muscle. Heat shock proteins (HSPs) are an important family of endogenous, protective proteins. HSP90 and HSP70 families show elevated levels under beat stress. Mechanical stress, such as physical exercise, is known to induce not only muscular hypertrophy but also the elevation of HSPs expression in skeletal muscle. The purpose of this study was to determine whether heat stress facilitates the stretch-induced hypertrophy of skeletal muscle cells. Cultured rat myotubes (L6) were plated on collagenized Silastic membranes and incubated at 41 degrees C for 60 and 75 minutes (heat shock). Following the incubation, the cells were subjected two-second stretching and four-second releasing for 4 days at 37 degrees C. Protein concentrations in the homogenates and pellets of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. The protein concentration of cells following mechanical stretching following heat shock was significantly higher than that following either heat shock or mechanical stretching alone. HSP72 in supernatants and HSP90 in pellets increased under heat shock and/or mechanical stretching. HSP90 in supernatants decreased following heat shock and/or mechanical stretching. Changes in HSPs and cellular protein concentrations in stressed cells suggest that the expression of HSPs may be closely related with muscular hypertrophy.     

Effects of chronic portal hypertension on small heat-shock proteins in mesenteric arteries

Previous studies have shown that impaired vasoconstrictor function in chronic portal hypertension is mediated via cAMP-dependent events. Recent data have implicated two small heat-shock proteins (HSP), namely HSP20 and HSP27, in the regulation of vascular tone. Phosphorylation of HSP20 is associated with vasorelaxation, whereas phosphorylation of HSP27 is associated with vasoconstriction. We hypothesized that alterations in the expression and/or phosphorylation of small HSPs may play a role in impaired vasoconstriction in portal hypertension. A rat model of prehepatic chronic portal hypertension was used. Studies were conducted in small mesenteric arteries isolated from normal and portal hypertensive rats. Protein levels of HSP20 and HSP27 were detected by Western blot analysis. Protein phosphorylation was analyzed by isoelectric focusing. HSP20 mRNA expression was determined by RT-PCR. To examine the role of cAMP in the regulation of small HSP phosphorylation and expression, we treated both normal and portal hypertensive vessels with a PKA inhibitor Rp-cAMPS. We found both an increased HSP20 phosphorylation and a decreased HPS20 protein level in portal hypertension, both of which were restored to normal by PKA inhibition. However, PKA did not change HSP20 mRNA expression. We conclude that decreased HSP20 protein level is mediated by cAMP-dependent pathway and that impaired vasoconstrictor function in portal hypertension may be partially explained by decreased expression of HSP20. We also suggest that the phosphorylation of HSP20 by PKA may alter HSP20 turnover.     

Endogenous expression and developmental changes of HSP72 in rat skeletal muscles

The purpose of the present study was to determine whether endogenous factor(s) contributes to the expression of heat shock proteins (HSPs) during the early developmental stages of rat skeletal muscles. HSP72 was expressed in both the soleus and plantaris muscles at embryonic day 22 (E22). On the basis of myosin heavy chain (MHC) immunohistochemistry, HSP72 was specifically expressed in slow type I fibers in both muscles. These slow fibers were observed throughout the entire cross section of the soleus muscle and only in the deep region (close to the bone) of the plantaris muscle. These results indicate that the expression of HSP72 is related to endogenous factors associated with type I fibers, because E22 rats have minimal exogenous influences and the soleus and plantaris muscles of E22 rats have similar metabolic and contractile profiles at this stage of development. We then examined the changes in HSP72 and heat shock cognate (HSC) 73 in the same two muscles from E22 to postnatal day 56 via Western blotting. The level of HSP72 in the soleus muscle gradually increased in parallel with the increment in the type I MHC isoform. Compared with the soleus, only a small amount of HSP72 could be detected in the plantaris muscle throughout the developmental period. For both muscles, HSC73 reached levels observed in adult muscles at postnatal day 3, and these levels were unchanged thereafter. These results indicate that the expression of HSP72, but not HSC73, is influenced by both endogenous and exogenous factors during the embryonic and early developmental periods.     

HSP90, HSP70, and GAPDH directly interact with the cytoplasmic domain of macrophage scavenger receptors.

The macrophage scavenger receptor (MSR) is a trimeric membrane protein which binds to modified low-density lipoprotein (LDL) and has been indicated in the development of atherosclerosis. It has recently been demonstrated that the N-terminal cytoplasmic domain of MSR has an important role in the efficient internalization and cell-surface expression of the receptor. This study shows that the N-terminal cytoplasmic domain in bovine was constructed using a peptide architecture technique in which the peptide chain was bundled at their C-terminus to yield a trimeric form and that this did not form an ordered structure. Furthermore, the binding proteins to the cytoplasmic domain of MSR were determined for the first time using a peptide affinity column. Sequence analyses of the specific binding proteins in bovine revealed that heat shock protein 90 (HSP90), heat shock protein 70 (HSP70), leucine aminopeptidase (LAP), adenocylhomocysteinase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were included. GST-pull-down assay and immunoprecipitation analyses on HSP90, HSP70, and GAPDH showed that all these proteins could bind to the cytoplasmic domain of MSR in vitro and in vivo. These proteins interact with the cytoplasmic domain directly and may have an effect on the functions of MSR such as internalization, cell-surface expression, and signal transduction.