The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation.

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.

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.

Role of the small heat shock proteins in regulating vascular smooth muscle tone.

BACKGROUND: Vasospasm occurs in conduits used for vascular reconstructions. The small heat shock proteins, HSP20 and HSP27, coordinately regulate vascular smooth muscle tone. Phosphorylated HSP20 is associated with vasorelaxation, and phosphorylated HSP27 inhibits the phosphorylation of HSP20 and relaxation. We hypothesized that the relationship between the phosphorylated states of these two proteins might dictate the tone of a vessel and may contribute to vasospasm. STUDY DESIGN: Sodium nitroprusside relaxation of vascular smooth muscle was recorded using pig coronary artery and human saphenous vein. Segments were frozen and homogenized, and extracted proteins were separated by one- and two-dimensional gel electrophoresis, transferred to Immobilon (Millipore), and probed with anti-cGMP-dependent protein kinase (anti-PKG), -HSP20, -HSP27, and -phosphoHSP27 antibodies. Band intensity was estimated using densitometry. RESULTS: Pig coronary artery completely relaxed (100%) with SNP (10(-7)M), but human saphenous vein only partially relaxed (20%). The levels of cGMP-dependent protein kinase and HSP20 were similar in the two tissue types. Human saphenous vein had significantly higher levels of HSP27 versus pig coronary artery (30.14 +/- 0.8 versus 6.62 +/- 0.2 pixels/mg; p < or = 0.001) and phosphoHSP27 (8.29 +/- 3.43 versus 0.012 +/- 0.008 pixels/mg; p < or = 0.001). CONCLUSIONS: Human saphenous vein contained significantly higher levels of HSP27 and pHSP27. Increased levels of phosphorylated HSP27 might contribute to vasospasm in human saphenous vein.

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.

Transducible recombinant small heat shock-related protein, HSP20, inhibits vasospasm and platelet aggregation.

BACKGROUND: Human saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstruction. Injury during harvest leads to vasospasm and a thrombogenic endoluminal surface. A proteomic transduction approach was developed to prevent vein graft vasospasm and thrombosis. METHODS: Recombinant HSP20 protein linked to the TAT protein transduction domain was generated in a bacterial expression system (TAT-HSP20). The effect of this protein on the inhibition of smooth muscle contraction was determined using rings of rabbit aorta and HSV in a muscle bath. In addition, the effects of TAT-HSP20 on platelet aggregation were determined in vitro using human citrated whole blood. RESULTS: Recombinant TAT-HSP20 inhibited norepinephrine-induced contraction of rabbit aortic and HSV segments. Similarly, TAT-HSP20 induced smooth muscle relaxation in HSV segments precontracted with norepinephrine. In human-citrated whole blood, platelet aggregation was significantly inhibited by TAT-HSP20 in a dose-dependent manner. CONCLUSIONS: The results of this study demonstrate that recombinant TAT-HSP20 inhibits vascular smooth muscle contraction and platelet aggregation. This suggests that HSP20 may be an ideal effector molecule to target as a proteomic approach to enhance early vein graft patency rates by preventing acute vasospasm and thrombosis.

Transduction of peptide analogs of the small heat shock-related protein HSP20 inhibits intimal hyperplasia.

BACKGROUND: Human saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstructions. However, vasospasm can lead to early graft failure. The leading cause of delayed graft failure is intimal hyperplasia. OBJECTIVE: To develop a proteomic approach to prevent vein-graft spasm and intimal hyperplasia. METHODS: Biomimetic peptide analogs of the small heat shock-related protein HSP20, containing a protein transduction domain (PTD), a phosphorylated serine, and a sequence of HSP20 surrounding the phosphorylation site (PTD-pHSP20), or a scrambled sequence of the same amino acids surrounding the phosphorylation site (PTD-scHSP20) were synthesized. The peptides were used in muscle bath and organ culture experiments with human saphenous vein (HSV) segments. Cultured smooth muscle cell lines were used to determine the effect of the peptides on proliferation and migration. RESULTS: In HSV rings precontracted with norepinephrine, PTD-pHSP20 but not PTD-scHSP20 led to relaxation. There was no significant difference in smooth muscle cell proliferation in cells treated with PTD-pHSP20 compared with PTD-scHSP20. Treatment with PTD-pHSP20 significantly inhibited cellular migration compared with PTD-scHSP20. Control, untreated, and PTD-scHSP20-treated saphenous veins had significant increases in intimal thickness after culture. This intimal thickening was completely inhibited by treatment with PTD-pHSP20. CONCLUSIONS: Protein transduction of biologically active motifs of HSP20 can affect pathologic and physiologic responses of HSV and represents a novel proteomic-based therapeutic approach. CLINICAL RELEVANCE: We have been a part of the genomics era and are now viewing the emergence of "proteomics." The genome is linear and relatively easy to examine; however the proteome is much more complex and dynamic. In essence, the purpose of gene therapy is to manipulate the genome to produce a particular protein. This manuscript describes a new proteomic approach in which the biologically active part of a protein is directly introduced into vascular cells. Peptides were synthesized which contained a total of 24 amino acids, 11 of which represent a protein transduction domain or "carrier" while the other 13 are the biologically active "cargo." These synthetic peptides prevent spasm (contraction) and intimal hyperplasia in segments of human saphenous vein treated ex vivo. Preclinical development is currently underway to develop these molecules as a proteomic-based vein harvest solution to enhance vein-graft patency.

Inactivity-induced modulation of Hsp20 and Hsp25 content in rat hindlimb muscles.

Denervation decreases small heat shock protein (HSP) content in the rat soleus muscle; however, it is unknown whether this change is due to inactivity or absence of a nerve-muscle connection. Spinal cord isolation (SI) is a model of inactivity with an intact neuromuscular connection. After 7 days of SI, Hsp20 and Hsp25 levels in the soleus, plantaris, and adductor longus muscles were lower than in control rats, whereas Hsp20 was unchanged and Hsp25 increased in the tibialis anterior. The results for the soleus indicate that these small HSPs respond to inactivity and that this response is not influenced by neural activity-independent factors. Furthermore, the data indicate that these HSPs are impacted to a greater degree in muscles that are predominantly slow or have an antigravity function than in flexor muscles. Understanding the regulation of these HSPs during chronic reductions in neuromuscular activity may have valuable applications for conditions such as spinal cord injury.

Sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of the heat shock-related protein 20 (HSP20).

PURPOSE: Sildenafil is an oral phosphodiesterase type 5 inhibitor that is a vasodilator used in the treatment of erectile dysfunction. Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of the heat shock related protein 20 (HSP20). The purpose of this study was to determine if sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of HSP20. MATERIALS AND METHODS: Peptides containing an 11 amino acid enhanced protein transduction domain (PTD) and the functional 13 amino acid sequence of HSP20 with a phosphoserine (PTD-pHSP20) were synthesized using F-MOC technology. Rings of porcine coronary artery were suspended in a muscle bath and sub-maximally contracted with serotonin. Increasing concentrations of sodium nitroprusside (SNP; 0.01-10 microM), sildenafil (0.01-100 microM), or PTD-pHSP20 (0.1-1.0 mM) were added to the baths and the percent relaxation was recorded. To determine if sildenafil-induced vasorelaxation was associated with increases in the phosphorylation of HSP20, rings of porcine coronary artery were untreated (control) or treated with SNP (10 microM) or sildenafil (100 microM) for 2, 5, and 10 min and then snap frozen. Extracted proteins were then separated using two-dimensional SDS-PAGE, transferred to a membrane, and probed for HSP20. RESULTS: Sildenafil induced vasorelaxation of pre-contracted coronary artery in a dose-dependent manner. Sildenafil-induced vasorelaxation was associated with an increase in the phosphorylation of HSP20. Transduction of peptide analogues of pHSP20 led to a dose-dependent relaxation of pre-contracted porcine coronary artery. CONCLUSIONS: These findings suggest that sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of HSP20 and that transduction of phosphopeptide analogues of HSP20 is sufficient for relaxation of vascular smooth muscle.

Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.

Diets enriched in sucrose or fat increase gluconeogenesis and G-6-Pase but not basal glucose production in rats.

High-fat (HFD) and high-sucrose diets (HSD) reduce insulin suppression of glucose production in vivo, increase the capacity for gluconeogenesis in vitro, and increase glucose-6-phosphatase (G-6-Pase) activity in whole cell homogenates. The present study examined the effects of HSD and HFD on in vivo gluconeogenesis, the catalytic and glucose-6-phosphate translocase subunits of G-6-Pase, glucokinase (GK) translocation, and glucose cycling. Rats were fed a high-starch control diet (STD; 68% cornstarch), HSD (68% sucrose), or HFD (45% fat) for 7-13 days. The ratio of 3H in C6:C2 of glucose after 3H2O injection into 6- to 8-h-fasted rats was significantly increased in HSD (0.68 +/- 0.07) and HFD (0.71 +/- 0.08) vs. STD (0.40 +/- 0.10). G-6-Pase activity was significantly higher in HSD and HFD vs. STD in both intact and disrupted liver microsomes. HSD and HFD significantly increased the amount of the p36 catalytic subunit protein, whereas the p46 glucose-6-phosphate translocase protein was increased in HSD only. Despite increased nonglycerol gluconeogenesis and increased G-6-Pase, basal glucose and insulin levels as well as glucose production were not significantly different among groups. Hepatocyte cell suspensions were used to ascertain whether diet-induced adaptations in glucose phosphorylation and GK might serve to compensate for upregulation of G-6-Pase. Tracer-estimated glucose phosphorylation and glucose cycling (glucose <--> glucose 6-phosphate) were significantly higher in cells isolated from HSD only. After incubation with either 5 or 20 mM glucose and no insulin, GK activity (nmol. mg protein(-1). min(-1)) in digitonin-treated eluates (translocated GK) was significantly higher in HSD (32 +/- 4 and 146 +/- 6) vs. HFD (4 +/- 1 and 83 +/- 10) and STD (9 +/- 2 and 87 +/- 9). Thus short-term, chronic exposure to HSD and HFD increase in vivo gluconeogenesis and the G-6-Pase catalytic subunit. Exposure to HSD diet also leads to adaptations in glucose phosphorylation and GK translocation.

Elevated basal PI 3-kinase activity and reduced insulin signaling in sucrose-induced hepatic insulin resistance.

Sucrose feeding reduces the ability of insulin to suppress glucose production and hepatic gluconeogenesis. The present study examined the effect of a high-sucrose diet on early insulin-signaling steps in the liver. Rats were provided a high-starch (STD, control diet) or high-sucrose diet (HSD) for 3 wk. On the day of study, overnight-fasted rats were anesthetized and injected with either saline (n = 5/diet group) or insulin (2 mU/kg, n = 5/diet group) via the portal vein. Portal venous blood and liver tissue were harvested 2 min after injections. Portal vein plasma glucose levels were not significantly different among groups, pooled average 147 +/- 12 mg/dl. Western blot analysis revealed no significant differences in the amount of insulin receptor (IR), insulin receptor substrates-1 and -2 (IRS-1, IRS-2), and the p85 subunit of phosphatidylinositol (PI) 3-kinase. In contrast, the amount of the p110beta subunit of PI 3-kinase was increased approximately 2-fold in HSD vs. STD (P < 0.05). After saline injection, tyrosine phosphorylation (pY) of IR, IRS-1, and IRS-2 was not significantly different between groups. However, PI 3-kinase activity associated with phosphorylated proteins was increased approximately 40% in HSD vs. STD (P < 0.05). After insulin injection, pY of the IR was not different between groups, whereas pY of IRS-1 and IRS-2 was reduced (P < 0.05) in HSD vs. STD. In addition, association of IRS-1 and IRS-2 with p85 was significantly reduced in HSD vs. STD. These data demonstrate that an HSD impairs insulin-stimulated early postreceptor signaling (pY of IRS proteins, IRS interaction with p85). Furthermore, the increased amount of p110beta and increased basal PI 3-kinase activity suggest a diet-induced compensatory response.