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Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle¹

CHARLES R. FLYNN^*, PADMINI KOMALAVILAS^*,, DERON TESSIER, JEFFREY THRESHER, ERIC E. NIEDERKOFLER^|, CATHERINE M. DREIZA^*, RANDALL W. NELSON^|, ALYSSA PANITCH^*, LOKESH JOSHI^¶ and COLLEEN M. BROPHY^*,

^* Departments of Bioengineering,
Kinesiology, and
^¶ Plant Biology, Arizona State University, Tempe;
Carl T. Hayden Veterans Affairs Medical Center, Phoenix;
Mayo Clinic Scottsdale, Department of Surgery, Scottsdale; and
^| Intrinsic Bioprobes Inc., Tempe, Arizona, USA

²Correspondence: Department of Bioengineering, Arizona State University, Tempe, AZ 85287-9709, USA. E-mail colleen.brophy{at}asu.edu

SPECIFIC AIM

Activation of cyclic nucleotide-dependent signaling pathways leads to relaxation of vascular smooth muscle and increases in the phosphorylation of HSP20. The aim of this study was to use an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into intact strips of porcine coronary artery smooth muscle to examine the physiologic relevance of HSP20.

PRINCIPAL FINDINGS

1. Treatment of serotonin precontracted porcine coronary muscles with the phosphodiesterase inhibitor papaverine led to relaxation of the muscles and increases in phosphorylation of HSP20

2. The transduction of PTD-phosphopeptide analogs of HSP20 led to a dose-dependent relaxation of serotonin precontracted muscles

3. Peptides containing the protein transduction domain coupled to a random orientation of the amino acids in the phosphopeptide analog of HSP20 did not cause relaxation

4. The phosphopeptide analogs of HSP20 did not induce phosphorylation of endogenous HSP20, suggesting that peptide alone was sufficient to cause relaxation

5. Transduction of the PTD-phosphoHSP20 peptide led to relaxation of norepinephrine precontracted rabbit aortic smooth muscles

6. PTD-phospho HSP20 was effectively transduced into smooth muscle strips as well as 3T3 fibroblasts and demonstrated a diffuse distribution of peptides throughout the cell

Phosphorylation of HSP20 is associated with cyclic nucleotide-dependent vasorelaxation
Papaverine (a phosphodiesterase inhibitor) treatment of porcine coronary artery led to relaxation of serotonin precontracted muscles (Fig. 1 B). Phosphorylation of endogenous HSP20 was determined using mass spectrometric immunoassay (MSIA). With high mass accuracy (0.03%) and resolution (M/m 1000), MSIA enabled the detection and distinction of nonphosphorylated HSP20 (wild-type; 17,551 Da; Fig. 2 B, black and green traces) and the phosphorylated isoform of HSP20 (17,631 Da; Fig. 2B , blue trace). This vasorelaxation was associated with a mass shift in HSP20 of 80 Da, which correlates with the mass of the phosphate group. Previous experiments have demonstrated that increases in the phosphorylation of HSP20 are associated with cyclic nucleotide-dependent relaxation of other muscles.

View larger version (17K): [in this window] [in a new window]   Figure 1. Treatment of intact strips of vascular smooth muscle with phosphopeptide analogs of HSP20. Rings of porcine coronary artery were suspended in a muscle bath. A cumulative dose response to serotonin was determined by repeated additions of log molar increases of serotonin (A). Rings of porcine coronary artery were precontracted with serotonin (1 µM) for 10 min and cumulative increasing log molar doses of the phosphodiesterase inhibitor papaverine were added (B). Rings of porcine coronary artery were precontracted with serotonin (1 µM) for 10 min, then treated with cumulative doses (0.1–1 mM) of PTD-phosphoHSP20 (YARAAARQARAWLRRASpAPLPGLK, filled circles) or PTD scrambled phosphoHSP20peptides (YARAAARQARAPRKSpLWALGRPLA, open circles, C). % of relaxation is the force of the maximal serotonin contraction minus the force after the addition of the peptide divided by the force of the maximal serotonin contraction x 100 (n=5 for all 3 experiments, p denotes a phospho-serine, * = < 0.05 compared with scrambled peptide).

View larger version (30K): [in this window] [in a new window]   Figure 2. MALDI-MSIA spectra of extracts from arterial rings treated with phosphopeptide analogs of HSP20. Rings of porcine coronary artery were treated with serotonin (10-6 M) alone (A, black trace), serotonin followed by papaverine (10-3 M) (B, green and blue traces), or serotonin followed by the phosphoHSP20 peptide (A, green trace), homogenized, and the homogenate was analyzed using MSIA. Antigens were captured using anti-HSP20 affinity tips, eluted using a sinapinic acid/acetonitrile/TFA matrix solution and analyzed by MALDI-TOF MS. Three different MALDI-MS spectra are shown overlapped on the same axis. Peaks corresponding to antigens of known molecular mass are labeled. Inset: Higher magnification showing the masses of phosphorylated and nonphosphorylated HSP20 isoforms. Results are typical of 3 separate experiments.

Transduction of phosphopeptide analogs of HSP20 leads to dose-dependent relaxation of serotonin precontracted muscles
Peptides were designed that contained an enhanced protein transduction domain linked to a phosphopeptide analog of HSP20. Rings of porcine coronary artery were suspended in a muscle bath and precontracted with serotonin. Addition of the phosphopeptide analogs of HSP20 led to a dose-dependent relaxation of the muscles (Fig. 1C ). The addition of analogs that contained a protein transduction domain linked to a scrambled sequence of the phosphopeptide analogs did not induce relaxation (Fig. 1C ). Thus, protein transduction domains can be used to alter physiologic responses of intact vascular smooth muscles.

Mechanisms of vasorelaxation
The specific mechanisms by which the phosphopeptide analogs of HSP20 induce relaxation are not known. A likely possibility is that the phosphopeptide analogs could alter endogenous HSP20 phosphorylation by inhibiting phosphatases. However, there was no increase in the phosphorylation of endogenous HSP20 induced by the phosphopeptide analog (Fig. 2A ). This suggests that the analogs are inducing relaxation by some other mechanism. Another possible mechanism is that phosphorylated HSP20 reverses the pathways that activate muscle contraction (increases in intracellular Ca²⁺, activation of myosin light chain kinase and in phosphorylation of the regulatory myosin light chains). However, studies have demonstrated that cyclic nucleotide-dependent relaxation occurs under circumstances in which RMLC phosphorylation and oxygen consumption remain high. This suggests that cyclic nucleotide-dependent relaxation is independent of pathways involving cross bridge cycling. Additional untested mechanisms are that phosphorylated HSP20 mediates vasorelaxation through interaction with other small heat shock proteins, interaction with small GTP binding proteins, or a direct interaction with the actin cytoskeleton.

CONCLUSIONS AND SIGNIFICANCE

Results from this study suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles and that small peptide sequences (motifs) can mimic the effects of the entire HSP20 molecule. Results also indicate that the motif surrounding the phosphorylation site (serine 16) is physiologically relevant and that the phosphopeptide alone is sufficient to cause relaxation, likely having a direct effect on the target of phosphorylated HSP20. These data provide direct evidence that phosphorylated HSP20 may be an integral part of the signaling pathway that induces relaxation (Fig. 3 ).

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic of vasorelaxation. In response to a variety of stimuli, endothelial cells (EC) produce nitric oxide (NO). NO is a gaseous molecule that diffuses across the cell membrane, activating guanylyl cyclase (GC) in the neighboring smooth muscle cell (SMC). This leads to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). Forskolin leads to relaxation via another cyclic nucleotide-dependent signaling pathway, activation of adenylyl cyclase (AC) and increases in cAMP. This leads to activation of cAMP-dependent protein kinase (PKA). PKA and PKG phosphorylate the small heat shock-related protein, HSP20, which interacts with the actin cytoskeleton leading to vasorelaxation. Pharmacologic agents that are currently in use (italics) lead to vasorelaxation by activating adenylate cyclase (prostaglandins, PG’s), guanylate cyclase (sodium nitroprusside) or by inhibiting phosphodiesterases (cilistazol, papaverine, viagra).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1028fje; doi: 10.1096/fj.02-1028fje

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