ERK1/2; Akt

H-Ile-Lys-Val-Ala-Val-OH (0-2.5 mM, 0-48 h) stimulates bone marrow mesenchymal stem cell (BMMSC) proliferation and proliferating cell nuclear antigen (PCNA) production [2]. H-Ile-Lys-Val-Ala-Val-OH (5 mM, 24 h) initiates the BMMSC cell cycle, causing the cells to enter the S phase from G0/G1 and preventing them from entering the G2/M phase [2]. H-Ile-Lys-Val-Ala-Val-OH (0-2.5 mM, 0-48 h) activates the MAPK/ERK and PI3K/Akt signaling pathways by raising the phosphorylation levels of ERK1/2 and Akt in BMMSCs[2].

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IKVAV peptide was found to induce proliferation and proliferating cell nuclear antigen (PCNA) synthesis of BMMSC in a dose- and time-dependent manner. Cell cycle analysis showed that the proportion of IKVAV-treated BMMSC in S phase in was higher than controls. Western blot results suggested that mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathways were activated by IKVAV by enhancing phosphorylation levels of ERK1/2 and Akt in the BMMSCs. Meanwhile, phosphorylation levels of ERK1/2 and Akt were partially blocked by ERK1/2 inhibitor (PD98059) and Akt inhibitor (wortmannin), respectively. Conclusions: Our results demonstrated that IKVAV stimulated BMMSC population growth and proliferation by activating MAPK/ERK1/2 and PI3K/Akt signalling pathways. This study is the first to reveal an enhancement effect of IKVAV peptide on BMMSC at the signal transduction level, and the outcome could provide experimental evidence for application of IKVAV-grafted scaffolds in the field of BMMSC-based tissue engineering [2].

Laminin is a basement membrane glycoprotein that has diverse biological activities. A sequence on the A chain containing IKVAV (Ile-Lys-Val-Ala-Val) has been shown to promote neurite outgrowth, cell adhesion, and tumor growth and metastasis. Here we have determined the structural requirements of this synthetic peptide for biological activity. Twelve-amino acid-long all-L- (LAM-L) and all-D-peptide (LAM-D) segments as well as an alternating D- and L-amino acid-containing peptide (LAM-DL), which included the IKVAV sequence (residues 2097-2108), were synthesized. Circular dichroism spectral analysis revealed a mirror image conformation of LAM-D and LAM-L with mainly beta-sheet and to a minor extent alpha-helical structure. LAM-DL did not exhibit any significant ordered conformational features. LAM-D and LAM-L showed similar cell attachment activities for rat pheochromocytoma cells (PC12), whereas LAM-DL was inactive. A peptide analog with randomized IKVAV sequence (LAM-RM) was also inactive. A similar trend was observed in competition experiments of the four peptides with laminin in analogous cell attachment assays. In in vivo experiments, both LAM-D and LAM-L were capable of increasing tumor growth when subcutaneously injected into mice with murine melanoma cells B16F10. Results indicate that the conformational status of the IKVAV domain is a contributing factor in determining the biological activity but that there is no strict requirement for a specific chirality. There is a likely sequence specificity to the IKVAV region [1].

Cell Proliferation Assay[2]
Cell Types: BMMSC
Tested Concentrations: 0, 0.004, 0.02, 0.1, 0.5 and 2.5 mM
Incubation Duration: 0, 12, 24, 36, 48 h
Experimental Results: Stimulated proliferating cell nuclear antigen (PCNA) synthesis and induced proliferation of bone marrow mesenchymal stem cell (BMMSC) in a dose- and time-dependent manner.

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Cell Cycle Analysis[2]
Cell Types: BMMSC
Tested Concentrations: 5 mM
Incubation Duration: 24 h
Experimental Results: Induced the BMMSC cell cycle for cells to enter S phase from G0/G1 and arrested them from entering G2/M phase; the increased proportion of S phase in cell cycle was considered to be a sign of BMMSC proliferation.

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Western Blot Analysis[2]
Cell Types: BMMSC
Tested Concentrations: 0, 0.004 , 0.02, 0.1, 0.5 and 2.5 mM
Incubation Duration: 0, 12, 24, 36, 48 h
Experimental Results: Increased Dramatically the levels of p-ERK1/2 and p-Akt in a dose- and time-dependent manner.

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Cell viability assay [1]
Cell viability was examined using CCK‐8 assay. BMMSC (5 × 104/well) were cultured in 96‐well plates with αMEM medium containing 8% FBS. Twenty‐four hours later, medium was replaced with αMEM containing IKVAV or PBS (control group); treated period ranged from 0 to 72 h. Concentration gradient of IKVAV in medium ranged from 0.004 to 2.5 mm. For quantitative analysis of cell proliferation, 10 μl WST‐8 solution was added to each well. After the given treatment period, absorbance at 450 nm was monitored by ELISA. Cell proliferation was calculated by normalizing optical densities (OD) to those of control cells incubated in PBS.

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Flow cytometry analysis [1]
Cell cycle and apoptosis of BMMSC treated with IKVAV were determined by FCM analysis. Apoptosis was revealed using an annexin V‐FITC apoptosis detection kit according to the manufacturer's protocol. BMMSCs were cultured at 6 × 105 cells/ml in 6‐well plates, in αMEM containing IKVAV of various concentrations or PBS (control group) for 24 h. Cells were harvested by trypsinization, then washed twice in cold PBS then centrifuged at 700 g. In the region of 1 × 105 to 1 × 106 cells were resuspended in 500 μl binding buffer, centrifuged again at 1000 rpm for 5 min before the supernatant being removed. [1]
For cell cycle assay, −20 °C pre‐cooled 90% ethanol was added slowly to the cells, which were then resuspended and kept overnight in an ice bath. Cells were centrifuged again at 1000 rpm for 5 min and supernatant was removed. They were resuspended in 250 μl PBS with 2 μl RNaseA (1 mg/ml in deionized water) and kept in a 37 °C water bath for 40 min; 50 μl PI (100 μg/ml in PBS) was added and cells were stained in the dark for 20 min. The cell cycle was examined at 488 nm by flow cytometry and cell cycle proration was analysed by Cell Quest and Modfit software. [1]
For the assay of apoptosis, cells were resuspended in 500 μl binding buffer and transferred to a sterile flow cytometry glass tube. Five microlitres of annexin V‐FITC and 5 μl propidium iodide were added then incubated in the dark for 10 min, at room temperature. Cells were analysed by flow cytometry at 488 and 530 nm. Distribution of cells was analysed using Cell Quest™ software in the flow cytometer within 1 h of staining. Data from 10 000 cells were collected for each data file. Apoptotic cells were identified as being annexin V‐FITC‐positive and PI‐negative.

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Real‐time fluorescence quantitative polymerase chain reaction [1]
Total cell mRNA was extracted from IKVAV‐induced BMMSC using Trizol reagent, according to the manufacturer's instructions. Isolated RNA was stored at −70 °C in diethylpyrocarbonate (DEPC)‐treated water, and quantity and quality of RNA were determined by absorbance of SYBR green II fluorescent dye at 260/280 nm. Total RNA was reverse transcribed into complementary DNA (cDNA) using Revert Aid First Strand cDNA Synthesis Kit according to the manufacturer's instructions. Primer sequences used in this context are listed as follows: PCNA primers: sense, TTTCACAAAAGCCACTCCACTG; antisense, CTTTAAGTGTCCCATGTCAGCAAT. GAPDH was used as internal control and detected using the following primers: sense, CGCTAACATCAAATGGGGTG; antisense, TTGCTGACAATCTTGAGGGAG. Polymerase chain reaction (PCR) amplification was performed with the following parameters: 40 amplification cycles for PCNA and GAPDH (95 °C for 1 min, at 58 °C for 45 s, 72 °C for 20 s). Gene expression was presented using a modification of the 2−ΔΔCt method.

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Western blot analysis [1]
BMMSCs were treated with IKVAV at different concentrations in the presence or absence of kinase inhibitors, as indicated for a variety of time periods. Cells were treated with lysis buffer (1 mm phenylmethylsulphonylfluoride was added before using). Lysates were subsequently centrifuged at 12 000 g for 5 min and supernatant was collected for protein analysis; sample protein concentration was determined using the Bradford Protein Assay Kit. Equal amounts of protein from cell lysates were first resuspended in sample buffer containing 62 mm Tris‐HCl (pH 6.8), 2% sodium dodecylsulphate, 10% glycerol, 5% β‐mercaptoethanol and 0.04% bromphenol blue, then resolved by sodium dodecylsulphate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After brief washing in Tris‐buffered saline Tween‐20 (TBST) (25 mm Tris‐HCl (pH 7.5), 50 mm NaCl, 0.1% Tween‐20), membranes were blocked with 5% (in TBST) skimmed dried milk for 1 h. Membranes were incubated overnight at 4 °C with the appropriate concentration of primary antibody. After washing in TBST, membranes were incubated in secondary antibody for 30 min, washed again in TBST and left on a shaking table. Blots were detected using an ECL kit, and signals were quantified by scanning densitometry. All data were expressed as relative differences between control and treated cells after normalization to GAPDH expression. In addition, PD98059 and wortmannin were employed to inhibit MAPK/ERK1/2 and PI3K/Akt signals in the experiment. BMMSCs were pre‐treated with the appropriate inhibitor for 30 min then the IKVAV was added. Western blot analysis was performed 24 h following IKVAV treatment.

[1]. The all-D-configuration segment containing the IKVAV sequence of laminin A chain has similar activities to the all-L-peptide in vitro and in vivo. J Biol Chem. 1992 Jul 15;267(20):14118-21.

[2]. IKVAV regulates ERK1/2 and Akt signalling pathways in BMMSC population growth and proliferation. Cell Prolif. 2014 Apr;47(2):133-45.

To examine whether activation of the above two signalling pathways, which mediated BMMSC proliferation by IKVAV, was functionally involved in phosphorylation of ERK1/2 and Akt, the ERK1/2 and Akt signalling pathway inhibitors PD98059 and wortmannin were employed. Under treatment of inhibitors, p‐ERK1/2 and p‐Akt levels were both down‐regulated, and PCNA expression and cell viability were reduced, correspondingly. Therefore, we concluded that both PCNA mRNA biosynthesis of PCNA and proliferation activities of IKVAV‐induced BMMSCs were regulated by MAPK/ERK1/2 and PI3K/Akt signalling pathways. Our results indicate that inactivation of Akt and ERK1/2 signalling pathways clearly suppressed IKVAV‐induced BMMSC growth and proliferation. Akt and ERK1/2 signalling pathways play an important role in IKVAV‐induced BMMSC population growth and proliferation.
In summary, we identified the role of IKVAV peptide in regulation of BMMSC population growth and proliferation through activating Akt and ERK1/2 signalling pathways. These results demonstrate that IKVAV peptide stimulated activation of MAPK/ERK1/2 and PI3K/Akt cascades, and promoted mRNA biosynthesis of PCNA and proliferation of BMMSC. This is the first study to point out the modulation function of IKVAV peptide on BMMSC growth and proliferation at the signal transduction level. This outcome provides experimental evidence for the application of IKVAV‐grafted scaffolds in the BMMSC‐based tissue engineering field.[1]

C25H48N6O6

528.69

528.363

131167-89-0

131343

H-Ile-Lys-Val-Ala-Val-OH; L-isoleucyl-L-lysyl-L-valyl-L-alanyl-L-valine

IKVAV

White to off-white solid powder

1.1±0.1 g/cm3

856.6±65.0 °C at 760 mmHg

471.9±34.3 °C

0.0±0.6 mmHg at 25°C

1.511

1.27

7

8

17

37

775

6

CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O)N

XQQUSYWGKLRJRA-RABCQHRBSA-N

InChI=1S/C25H48N6O6/c1-8-15(6)18(27)23(34)29-17(11-9-10-12-26)22(33)30-19(13(2)3)24(35)28-16(7)21(32)31-20(14(4)5)25(36)37/h13-20H,8-12,26-27H2,1-7H3,(H,28,35)(H,29,34)(H,30,33)(H,31,32)(H,36,37)/t15-,16-,17-,18-,19-,20-/m0/s1

(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S,3S)-2-amino-3-methylpentanoyl]amino]hexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-3-methylbutanoic acid

131167-89-0; H-Ile-Lys-Val-Ala-Val-OH; IKVAV; Ile-lys-val-ala-val; Isoleucyl-lysyl-valyl-alanyl-valine; L-Valine,L-isoleucyl-L-lysyl-L-valyl-L-alanyl-; (2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S,3S)-2-amino-3-methylpentanoyl]amino]hexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-3-methylbutanoic acid; SCHEMBL180642;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 100 mg/mL (189.15 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)