Regulation of Osteoblast Differentiation by Affinity Peptides of TGF-Î²1

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Tissue Engineering and Regenerative Medicine

Regulation of osteoblast differentiation by affinity peptides of TGF-#1 identified via phage display technology Yuhua Sun, Jing Tan, Xianzhen Yin, Baohua Wu, and Bo Feng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00492 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 9, 2018

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ACS Biomaterials Science & Engineering

Regulation of osteoblast differentiation by affinity peptides of TGF-β1 identified via phage display technology Yuhua Sun1, Jing Tan2, Xianzhen Yin1, Baohua Wu1, Bo Feng1* 1

Key Laboratory of Advanced Technology for Materials (Ministry of Education), School

of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China 2

School of Life Science, Shanxi Datong University, Datong 037009, China

*Corresponding author: Tel: +86 028 87634023; Fax: +86 28 87601371. E-mail addresses: [email protected] (Bo Feng)

ABSTRACT Transforming growth factor β1 (TGF-β1) plays a dual role in bone formation. In addition to promoting early differentiation of osteogenesis, it may also lead to uncontrolled extracellular matrix synthesis, inhibition of bone mineralization in the late stage, and aberrant bone remodeling. In this work affinity peptides of TGF-β1 (Tβms) were identified from a phage display library to modify the TGF-β1 signal transduction. Tβms with more order and compact structures tended to have a higher affinity to TGF-β1, but maintained a greater immunereactivity of TGF-β1. Tβms promoted the early osteoblast proliferation and had a negligible effect on the osteoblast differentiation. In synergy with exogenous TGF-β1, Tβms reduced the alkaline phosphatase (ALP) mRNA expression, but significantly improved the expression of osteocalcin (OCN), along with impaired phosphorylation of Smad2/3. Moveover, osteoblasts showed an overall increase in ALP activity and Ca deposition than the blank control. These results demonstrated that Tβms could weaken the inhibition of TGF-β1 on osteogenic differentiation in the late stage. Depending on the impact features of Tβms on TGF-β1 response, these peptides may help to modify the

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implant surfaces to optimize the bone remodeling of interface, and be of interest in design of multidomain peptides. Keywords：：TGF-β1; Affinity peptides; Osteoblast differentiation; Smad pathway

INTRODUCTION In the biomimetic ECM modification of biomaterial surfaces, the introduction of bioactive molecules can induce specific physiological responses of cells. Transforming growth factor β1 (TGF-β1) is a bidirectional cytokine, which is ubiquitous in vivo. It influences various biologic processes, such as inflammatory response, angiogenesis and wound healing 1-3. In most fibrotic processes and skeletal diseases, TGF-β1 represents a pivotal factor 4-8. In bone system, it helps to maintain the bone dynamic balance by controlling both osteoblast and osteoclast differentiation 9-10. TGF-β1 is secreted as a latent form and deposits in the bone matrix. During the bone resorption, it is released and activated. Through interacting with specific TGF-β receptors (TβR) on cell surfaces, TGF-β1 triggers the intracellular signaling cascade, leading to the phosphorylation of R-Smad proteins. Phosphorilated Smad2/3 (p-Smad2/3) forms a complex with Smad4 and translocates into nucleus to control target gene expression, initiating normal or pathological responses. The abnormal expression of TGF-β1 in bone remodeling stage will lead to the imbalanced bone formation and bone resorption. The mice with TGF-β1 gene knockout showed significant decreased bone remodeling after 4 weeks

11

. Almost no osteoblasts were found in the trabecular bone

12

.

However, in multiple myeloma (MM) patients, where expression and activation of TGF-β1 was enhanced, it helped to create a MM niche for enhancement of MM growth and acceleration of bone loss 13. It is reported that TGF-β1 promotes the osteoblast recruitment, proliferation and differentiation in the early stage, but inhibits progression of osteoblast differentiation 1, 14-15. Runx2 (Runt-related gene 2) is known as the major transcription factor controlling 2 ACS Paragon Plus Environment

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osteoblast commitment and differentiation 16-17. Smad3, activated by TGF-β1, can bind and repress Runx2 function to inhibit osteoblast differentiation

16, 18-19

. Smad3 mediates the

interaction of histone deacetylases (HDACs) with Runx2, resulting in the deacetylation of osteocalcin (OCN) promoter and further suppression of the OCN expression. Moreover, inhibitory Smads (Smad6 and Smad7) induced by the TGF-β1 signaling can trigger the repressive effect on osteogenic differentiation through the Smad ubiquitin regulatory factor 2 (Smurf2)-mediated Runx2 degradation

20-21

. With the in-depth exploration of TGF-β1

roles in various skeletal diseases, more new evidence shows that TGF-β1 contributes to the development of osteoarthritis and vicious cycle of bone metastasis

7, 22-25

. Blockage or

modulation of TGF-β1 signaling is considered as an effective approach of treating TGFβ-mediated pathologies 7, 13, 22, 26. Knockout of the TGF–β type II receptor (TβRII) 22, 25 and inhibitors of TGF-β type I receptor kinase

13, 24

could potently enhance osteoblast

differentiation, which is suppressed by high level of active TGF-β1. Antibodies against integrin-mediated TGFβ activation is also a promising way in treating organ fibrosis and tissue remodeling 5. Dotor et al. 27 have applied the TGF-β1 inhibitory peptides, which were identified from the phage display library, to reduce the fibrosis in injury of mice liver. The TGF-β1 inhibitor peptide (P17) binds TGF-β1 and blocks its interaction with TβRII. These results encourage us to find a peptide aimed at decreasing the inhibitory effect of TGF-β1 on progression of osteoblast differentiation during the osteointegration. Phage display technology makes it accessible to isolate affinity peptides of molecules interested. It has been widely used in the simulation of stuck agent, exciting agent, peptide vaccines, drugs research and other fields 28-30. In this work, a number of peptides with affinity to TGF-β1 (Tβms) were identified. After analyzing the sequence characteristics, we evaluated their affinity to TGF-β1, and investigated their effects on osteoblast proliferation and differentiation. The selected Tβms 3 ACS Paragon Plus Environment


showed an impact on TGF-β1 signaling by adjusting the phosphorilation of Smad2/3. As a result, they made a regulation on the osteoblast function. In comparison, affinity peptide Tβm16 (SGVYKVAYDWQH) may be more favorable for osteoblast differentiation.

EXPERIMENTAL PROCEDURES Affinity peptides biopanning. Recombinant human TGF-β1 (rhTGF-β1, PeproTech) was reconstituted in 10 mM citric acid (pH 3.0) to a certain concentration. Polystyrene 96-well plate was packaged by 50 µL rhTGF-β1 and NaHCO3 solution (0.1 M, pH 8.6, as Negative control) at 4 ºC overnight, respectively. Then the coating solution was abandoned absolutely and 150 µL BSA blocking buffer (Tris buffered saline (TBS, pH 7.5) containing 0.1% bovine serum albumin (BSA) and 0.1% Tween-20) was added. After 2 h the plate was washed with TBST (TBS with 0.1% Tween-20) 6 times. Phages of 1.5×1011 pfu/mL (plaque forming units (pfu) per milliliter) from the Ph.D.™ phage display library (New England Biolabs) were allowed to interact with the target molecules. Each of them expresses a 12-mer random peptide at the N-terminus of phage minor protein pIII. Unbound phages were washed away with TBST, while the bound phages were eluted by 100 µL of 0.2 M glycine-HCl (pH 2.2). These phages were then amplified in 200 µL Escherichia coli strain ER2738 (E. coli, New England BioLabs). The titration of phage was determined with the LB/IPTG (isopropyl-β-D -thiogalactosidase) /Xgal (5-bromo-4-chloro-3-indoyl-β-Dgalactosidase) agar plate. After three rounds of elution-amplification at decreasing concentrations of rhTGF-β1, the best binder phages were selected and cloned in E. coli, and then their DNA was extracted. The DNA region coding for their corresponding 12-mer peptide was sequenced. It was completed by the Beijing Zixi Biological Technology Company based on the ABI 3730XL sequencing platforms. The sequencing primers were -96 gIII sequencing primer (5´-HOCCCTCATAGT TAGCGTAACG-3´) and -28 gIII sequencing primer (5´-HOGTATGGGATTT TGCTAAACAAC-3´) purchased from New 4 ACS Paragon Plus Environment

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England BioLabs. DNAssist 2.2 was employed to convert the DNA sequence to a protein sequence. The basic physical properties of these binding-peptides were analyzed by ExPASy-ProtParam tool (http://web.expasy.org/protparam/). Peptide synthesis. The 12-mer peptides, identified through the phage displayed technology, were synthesized by ChinaPeptides Co., Ltd. via the solid phase method. Peptides were at least 90% pure as per HPLC. Molecular modeling studies. To model the affinity peptides, linear forms were built using molecular modeling software (Hyperchem 7.5, Gainesville, FL). To analyze the energy landscape and thus to achieve the minimum-energy structures, the chosen dihedral angles were first varied randomly, and then these newly formed initial peptide structures were minimized using the Polak-Ribiere conjugate gradient method. This process was reiterated until convergence of the gradient 0.01 kJ/mol was achieved using the Bio-CHARMM 27 force field. The lowest energy conformations were next solvated with water explicitly and, finally the overall system was energy minimized using the Polak-Ribiere conjugate gradient method by means of the convergence criterion and the force field parameters, as previously provided. The dimensions (x, y, z) of the box containing water and the peptides were 35×35×35 Å. The final molecular conformation was saved as picture. Affinity assay. To assess the specific antibody response to pIII fusion peptide, plaque reduction test was carried out. 10 µL peptide-displayed phage (P-Tβm) and 10 µL diluted rhTGF-β1 solutions were mixed and then incubated at 37 ºC for 1 h. Then the phage/rhTGF-β1 mixture was used to infect 200 µL E.coli culture by incubation at room temperature for 10 min. 3 mL melted top agar was added into the mixed solution and transferred to LB/IPTG/Xgal sugar plates. The binding of TGF-β1 to the displayed peptides at N-terminal domain of pIII will block the recognition of phage to the E.coli. If the phages 5 ACS Paragon Plus Environment


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fail to infect the E.coli, no phage plaques will be presented on the agar plates. The negative control was performed with phages only (no rhTGF-β1 added). The phage plaques produced on the plates were counted. The value of phage reduction was determined as: [(pfu rhTGF-β1−pfu rhTGF-β1 added)

no

/ ( pfu no rhTGF-β1)] ×100%.

In addition, ELISA assay was performed to evaluate the affinity between the rhTGF-β1 and Tβms. Tβms were diluted into 20 µg/mL by coating solution for immobilization onto the 96-well plate. Each group was set up 4 parallel samples. After coating at 4 ºC overnight, the 96-well plate was blocked by BSA blocking buffer for 2 h. Then, rhTGF-β1 of 1 ng/mL was added to interact with the coated Tβms. The amount of the bound TGF-β1 was determined with a mouse TGF-β1 ELISA kit (NeoBioscience Technology Co., Ltd.) based on the instruction. The standard curve of TGF-β1 was made through diluting the rhTGF-β1 into 1 ng/mL, 0.5 ng/mL, 0.25 ng/mL, 0.125 ng/mL, 0.0625 ng/mL and 0.03125 ng/mL, respectively. Blank control was set up in each test. Blocking efficiency of TGF-β1 immunoreactivity. To evaluate the blocking efficiency of TGF-β1 immunoreactivity by Tβms, Tβms of 20 µg/mL were premixed with 1 ng/mL TGF-β1 at 4 ºC overnight. Meanwhile, as control, 1 ng/mL TGF-β1 and 20 µg/mL Tβms were prepared separately. Each group had 3 parallel samples. The concentration of TGF-β1 was detected by a mouse TGF-β1 ELISA kit. Blocking efficiency = (C

rhTGF-β1−

CTGF-β1+Tβms−CTβms) / C TGF-β1. Cell culture. Osteoblasts were extracted from the sprague-dawley (SD) rat’s calvarial bone. The operation was as follows: the SD rat was put to death in 75% ethanol solution. After the alcohol cotton disinfection, open the head skin and extract the calvarial bone. The periosteum and surrounding connective tissue on the calvarial bone were removed until the bone was translucent. Remove the bone sutures, and clean the remaining bone with PBS. In another new culture dish, the remaining bone were cut into pieces, and then collected 6 ACS Paragon Plus Environment

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into the culture flask by the culture medium containing 10% newborn bovine serum (Abcam) and 90% α-MEM medium (Hyclone). They were cultured at 37 ºC in a 5% CO2 atmosphere. The experimental protocol was approved by the Institutional Animal Ethics Committee of the Southwest Jiaotong University. Cells of passages 3-5 were detached by 0.25% trypsin digestion (Trypsin-EDTA, Hyclone) and seeded onto the 96-well plate. Each group had four parallel tests. Each well contained 8000 cells. 24 h later, the culture medium was replaced by medium with 10 µg/mL, 20 µg/mL and 50 µg/mL Tβms. It was changed every two days. Cell activity was measured by an alamar blue method at 2 d, 4 d and 6 d. The alkaline phosphatase (ALP) activity was tested after 4 days and 6 days by ALP microplate test kit (Nanjing Jiancheng Bioengineering Institute). The cells were lysed in 1%Triton X-100 solution by repeated freezing and thawing. The relative ALP activity was obtained as the changed ALP concentration values (mg/mL) divided by the total protein content (mgprot/mL), which was measured with an enhanced Bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology). In addition, active TGF-β1 in the supernatant at 6 d was measured by a mouse TGF-β1 ELISA kit. To explore whether the Tβms modify the effect of a single dose of TGF-β1, osteoblasts were cultured in media supplied with 2 ng/mL rhTGF-β1 and 20 µg/mL Tβms. ALP activity was tested after 4 days and 6 days. Cells cultured for 6 days and 10 days were rinsed with water and treated with 200 µL 0.5 M HCl to dissolve the deposited calcium (Ca). The Ca concentration was detected by a Ca test kit (Nanjing Jiancheng Bioengineering Institute). The relative Ca deposition was obtained as the Ca2+ concentration values (mM/mL) divided by the total protein content (mgprot/mL). For calcium nodule staining, cells culturing for 10 days were fixed in 4% paraformaldehyde at 4 ºC for 15 min and then stained with 0.2% alizarin red at pH 5.0 for 15 min. The staining images were collected with optical microscope. 7 ACS Paragon Plus Environment


Real-time PCR analysis (RT-PCR). Total RNA of osteoblast cultured for 6 days and 10 days was extracted with RNAiso Plus reagent (TaKaRa) for RT-PCR detection. Reverse transcription process was conducted according to the manufacturer’s instructions of PrimeScriptTM RT Master Mix (TaKaRa). RT-PCR primers (Table S4) were designed based on cDNA sequences from the Ensembl database (http://asia.ensembl.org/index.html). They were synthesized by TsingKe Biological Technology. The targeted mRNA expressions were detected on the CFX96 Real-Time detection System (Bio-rad) with the SYBR® Premix Ex TaqTMⅡ(TaKaRa). Each group was performed in triplicate. The mean cycle threshold (Ct) value of each target gene was normalized against the Ct value of β-actin. The results of the expression of various genes are shown as fold change of the gene expression relative to the TGF-β1 group, and the 2−∆∆Ct method was applied. Western blot analysis. Osteoblasts cultured for 6 days and 10 days were washed with cold PBS buffer, lysed with RIPA lysis buffer (Beyotime Biotechnology) supplied with 1% 100 mM phenylmethanesulfonyl fluoride (PMSF), and then kept on ice for 30 min. Sonicate for 10~15 s to complete cell lysis and shear DNA. The cell lysate was centrifuged at 12000 g for 10 min at 4 ºC. Total protein of supernatants was determined by the BCA protein assay and unified to the same concentration with lysis buffer. Lysate was diluted at a ratio of 3:1 with protein loading buffer (4×, containing 2.5% β-Mercaptoethano) and heated at 65 °C for 18 min. Protein extracts were separated on a 5% concentration sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel and 10% separation gel at 100 V for 2 h, and then blotted onto polyvinylidene fluoride membrane (PVDF, Bio-Rad) for 2 h at 100 V (Mini-PROTEAN® Tetra Cell, Mini Trans-Blot® Module, and PowerPac™ Basic Power Supply, Bio-Rad). Prestained molecular weight marker (10 µl/lane, ten bands, 10 KD-180 KD, Beyotime Biotechnology) was loaded to determine the molecular weight. The PVDF membrane with targeted molecules was blocked for 2 h with 5 % BSA in TBST. Next, it 8 ACS Paragon Plus Environment

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was incubated overnight with a primary antibody at 4 °C. After washed with TBST, it was subsequently incubated with a secondary antibody conjugated with horseradish peroxidase (HRP). The bands were visualized by adding Clarity™ Western ECL Substrate (Bio-Rad). The images were acquired with a Molecular Imager® ChemiDocTM XR+ Imaging System with Image LabTM Software (Bio-Rad). The phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (1:1000, 52, 60 KD) and GAPDH (1:1000, 37 KD) antibodies, and the secondary antibody conjugated with HRP (1:1000) were bought from Cell Signaling Technology. Statistical analysis. All results are presented as mean ± SEM. The statistical analysis was performed in SPSS Statistics 17.0 using one-way or two-way ANOVA method. The significance was indicated at p value of less than 0.05.

RESULTS Identification of affinity peptides of TGF-β1. To identify affinity peptides of TGF-β1 (Tβms), we used a phage-displayed random 12-mer peptide library. After three cycles of biopanning-elution-amplification at decreasing concentrations of rhTGF-β1, a total of 25 phage plaques were obtained to sequence. The ratios of output to input of phages in each round were shown in Table S1. In terms of the phage recovery, there was only a weak enrichment. Although, in the biopanning the rhTGF-β1 concentration was gradually deceased, the purpose of the biopanning was to obtain phages with high affinity to TGF-β1 rather than high enrichment. Analysis of the Tβm sequences. 17 different peptides with affinity to TGF-β1 were identified (shown in Table 1). Affinity of ligands to the macromolecules firstly needs the “keyhole” associated “key” structure in 3D conformation. The “keyhole” structure in the molecules is usually formed by the infoldings of the hydrophilic amino acid residues driven by the hydrophilic interaction. It means that the ligands are necessary to have certain 9 ACS Paragon Plus Environment


hydrophilic properties. Most of the Tβms in Table 1 had medium hydrophilic property (−1.5 < GRAVY < 0.2). Ligands with too strong hydrophobicity are hard to dissociate to form electrostatic and hydrogen bonding interactions with the target, while ligands with too weak hydrophobicity are not easy to approach the affinity sites. Because the C terminal of the peptides is fixed on the phages, the amino acid composition near the N terminal is more important to the affinity

27

. It was noted that the amino acid fragments at the N terminal

usually had the amino acid residues H and S, or hydrophobic fragment, such as Tβm12. In addition, most of Tβms displayed net positive charges, and more than half had the theoretical isoelectric point (pI) > 7.4. Analysis of the amino acid distribution (Figure S1) showed that non-polar amino acids (leucine (L), proline (P), phenylalanine (F)), polar amino acids (tyrosine (Y), tryptophan (W), serine (S), threonine (T)), acidic amino acid (asparic acid (D)) and basic amino acids (histidine (H), lysine (K)) appeared more frequently than the overall peptide library. Especially, H displayed nearly three times over expected. H has positive charge and is also a strong hydrogen donor. Besides, there were 13 kinds of Tβms containing the P in their sequences. Its existence could assist the peptides to form different conformation, and thereby forming different recognition to the target molecule. TGF-β1 is generally secreted and stored in latent forms. Once TGF-β1 is released from its latent complex, it can bind to its receptors or other matrix proteins. It is reported that, in addition to the latency associated polypeptide (LAP), small proteoglycan decorin, α2-macroglobulin and neutralizing antibody (such as 4KV5) can bind to TGF-β1 and bar its access to the receptors

31

. In the Table S2, we compared the Tβm sequences with TGF-β

related sequences: TGF-β1 receptors (TβR-I, II, III) and ligand binding traps (LAP, decorin, α2-macroglobulin and 4KV5). These peptide sequences were acquired from the NCBI database. Results indicated that 14 kinds of Tβms had sequence similarity to the ligand 10 ACS Paragon Plus Environment

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binding traps. Three were similar to TβR-III. TβR-III can mediate the endocytosis of TGF-β1/TβR-II/TβR-I complex, thereby inhibiting the TGF-β-Smad signaling pathway. Affinity assay of the Tβms. According to the sequences acquired from the NCBI database, the theoretical isoelectric point (pI) value of of TβR-I and TβR-II are 8.27 and 5.58, respectively. In the TGF signaling pathway, TGF-β1 firstly interacts with TβR-II on the cell membrane, and then binds to the TβR-I to form a heterotetrameric TβR-II/ TβR-I complex. The pI value of TGF-β1 is 8.59. Based on the pI value, five Tβms with pI near to 5.58 were selected for the next experiment. They were Tβm2, Tβm4, Tβm9, Tβm14, and Tβm16, compared in detail in Table S3. These five peptides had some differences in sequence structure, similarity and hydrophobicity. Plaque reduction test is a method to estimate whether the antibody response is specific against the peptide displayed on pIII protein of phages

32

. N-terminal domain of pIII is

responsible for guiding the infection of E.coli by phage via attachment to the tip of the pilus. If TGF-β1 interacts with the displayed peptides, this domain will be blocked and phage will fail to infect the E.coli. Hence, the phage plaques will not be present on the agar plates. The images of agar plates were shown in Figure S2. The number of phages was determined by counting the number of plaques in a given plate. In Table 2, it can be seen that after interacting with TGF-β1, the amount of phages decreased. Moreover, with the increase of the TGF-β1 concentration, the value of phage reduction became larger. The phage displaying peptide Tβm16 (P-Tβm16) had the highest affinity to TGF-β1, while P-Tβm4 had the least. ELISA assay in Figure 1 showed the similar results. It was that more TGF-β1 interacted with Tβm16 and Tβm14 while less interacted with Tβm4. The reason was attributed to the amino acid distribution, sequence diversity and protein conformation. The prediction results of secondary structures in Figure 2 and Table S3 by DNASTAR 7.1 software using Gamier-Robson method revealed significant conformational differences 11 ACS Paragon Plus Environment


between these affinity peptides. Tβm16 possessed the most β-strand structure, while the other peptides all had the β-turn and coil structures. Tβm14 was the only peptide that had the α-helix structure. The structural models of these peptides in Figure 2 showed that Tβm16 and Tβm14 had more compact structures than the other three peptides. Effect of Tβms on osteoblast proliferation and differentiation. Osteoblasts were cultured with five different Tβms (Tβm2, Tβm4, Tβm9, Tβm14, and Tβm16) at three kinds of concentrations (10 µg/mL, 20 µg/mL and 50 µg/mL). Cell viability in Figure 3a and Figure S3a showed an increasement from 2 days to 6 days. Different Tβms displayed a significant difference in the OB viability (p < 0.05), and Tβms promoted the osteoblast proliferation in the first four days. At 2 d, significant difference was only observed at the different concentrations of Tβm16. At 6 d, osteoblast proliferation was higher at 50 µg/mL in the Tβm16 group, as was the case in Tβm9 and Tβm14 groups. However, osteoblasts in most Tβms groups showed a lower viability than the blank control. ALP activity assay on day 4 and day 6 showed that Tβms had little effect on ALP activity (Figure 3b and Figure S3b). Active TGF-β1 in the supernatant at 6 days was detected by an ELISA kit. Higher level of active TGF-β1 was present in Tβms groups, except for Tβm4, with concentration between 60 pg/mL and 110 pg/mL (Figure 3c and Figure S3c). Significant difference existed between the different Tβms (p < 0.05). The above-described observations suggested that Tβm types and concentrations indeed had an effect on the osteoblast proliferation. However, differences between groups were not always consistent. Synergistic effect of TGF-β1 and Tβms on osteoblast proliferation and differentiation. Based on above results, Tβm4, Tβm14 and Tβm16 at 20 µg/mL were selected to determine whether Tβms synergistically enhance or weaken osteoblast function with TGF-β1 signaling pathway activation. These groups were named as T+Tβm4, 12 ACS Paragon Plus Environment

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T+Tβm14 and T+Tβm16, respectively. The selection of TGF-β1 concentration (2 ng/mL) was according to some studies

33-34

, in which TGF-β1 of 2 ng/mL inhibited the OCN

expression. Firstly, the blocking efficiency of TGF-β1 immunoreactivity by Tβm4, Tβm14 and Tβm16 was evaluated by ELISA assay. Results in Figure 4a indicated that the TGF-β1 retained the highest immunoreactivity after interacting with Tβm16, followed by Tβm14 and Tβm4. Tβm4 had the sequence similarity to decorin up to 83.3%, which may lead to the strong blockage (~39.7%) of the binding of TGF-β1 to its antibody. Cell viability assay in Figure 3b suggested that rhTGF-β1 was not beneficial for the cell proliferation. Osteoblasts at 6 d showed higher viability in the blank group (Figure 4b and Figure S4a). The groups with TGF-β1 presented higher ALP activity at 4 d and 6 d (Figure 4c and Figure S4b). Among them, T+Tβm4 reduced the ALP activity, compared to the TGF-β1 group. Then, the expression of osteogenesis-related genes (ALP, COL I, OPN, OCN) and TGF-β1 pathway related genes (Runx2, TGF-β1, TβRII) were detected by RT-PCR at 6 d and 10 d (Figure 4d-e and Figure S4c-d). The expression of ALP begins in the early stage of osteoblast differentiation. COL I persists in early and mature osteoblasts

35

. OPN is

expressed in relatively immature osteoblasts, and OCN is expressed in mature osteoblasts, which appears at the mineralization stage

36-37

. Compared to the blank control, the mRNA

expression of Runx2, OPN, OCN and TGF-β1 were downregulated, while the ALP and COL I were upregulated in T+Tβm16 group, as the same in TGF-β1 and T+Tβm14 groups. The synergies of TGF-β1 and Tβms showed lower ALP level and higher OCN level than the TGF-β1 group. Among them, T+Tβm4 induced the significantly higher OCN expression, but lower expression of ALP, COL I and TβRII. For TGF-β1 group, the TβRII expression at



6 d was higher than that in the blank control but lower at 10 d, while the TGF-β1 expression showed an opposite pattern. To better understand the changes in gene expression in different groups, fold change of these genes were obtained by comparing the osteoblasts cultured for 10 days with 6 days (Figure S5). Runx2, ALP and COL I were downregulated in all groups from 6 days to 10 days, including the blank control. OPN and TβRII expression were most upregulated in T+Tβm4 group. The TGF-β1 group significantly upregulated the OCN expression, followed by the T+Tβm14 and T+Tβm16 groups. The expression and secretion of extracellular matrix, such as ALP, OPN and OCN, will strongly affect bone mineralization. Therefore, the Ca depositions were measured after cultured for 6 days and 10 days. Consequently, the groups with TGF-β1, especially the T+Tβm16 group, had more Ca deposition amount (Figure 3f). The Alizarin red staining of osteoblast mineralization at 10 d was showed in Figure S6. More calcium nodules formed in the T+Tβm16 group. In addition, protein levels of p-Smad2/3 were detected by western blot. TGF-β1 can mediate the signaling pathway in a Smad-dependent manner. Hence, p-Smad detection is a surrogate marker for TGF-β1 response. In Figure 5, the phosphorylation of Smad2/3 was downregulated in T+Tβm groups at 6 d, whereas enhanced in the T+Tβm14 and T+Tβm16 groups at 10 d, compared to the TGF-β1 group. On day 10 TGF-β1 group led to a significantly lower protein level of p-Smad2/3 than the blank control. As the downstream molecules of TGF-β1 receptor pathway, the difference in the protein levels of p-Smad2/3 was consistent with that in the TβRII mRNA expression in Figure 4d-e and Figure S4c-d. The Tβms may affect the osteoblast differentiation by tailoring the TGF-β1/ Smad signaling.


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DISCUSSION The results of this study demonstrated that affinity peptides (Tβms) of TGF-β1 could regulate osteoblast functions, suggesting its potential in modifing TGF-β1 effect in bone tissue regeneration and diseases that require control of the TGF-β1 active level. Tβms promoted the early osteoblast proliferation individually. Treated with TGF-β1, Tβms significantly increased the expression of osteoblast mineralization marker OCN with varied effects on the early osteoblast differentiation genes Runx2, ALP, COL I and OPN, depending on the interaction of Tβms with TGF-β1. Tβm16 improved the Ca deposition in the presence of exogenous TGF-β1. In this work, Tβms were firstly isolated from the phage display library via three rounds of biopanning. Analysis of the sequences revealed that most of the Tβms had medium hydrophilic property, basic or hydrophobic amino acid residues at the N-terminal, proline (P) in the sequences, and sequence similarity to the ligand binding traps (LAP, decorin, α2-macroglobulin and 4KV5 antibody). Then, five different peptides, specifically Tβm2, Tβm4, Tβm9, Tβm14 and Tβm16, were characterized in detail. Tβm16 had a high affinity to TGF-β1, but Tβm4 had the lowest affinity, despite exhibiting physicochemical properties similar to Tβm16. The difference in affinity may be a result of amino acid residue composition and conformational differences. The Tβm4 with more proline adopted a combination of coil, β-turn and β-strand structures, while the Tβm16 only had the β-strand structures (Figure 2). From the results of structure prediction, it was concluded that Tβms with more compact and order structures tended to have the higher affinity to TGF-β1. Then, the effect of Tβms on osteoblast function was studied. In bone formation four functional stages are involved: recruitment and proliferation of preosteoblast, bone matrix synthesis and maturation, mineralization and apoptosis or differentiation to osteocytes. 15 ACS Paragon Plus Environment


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During the proliferative phase (0-4 days), osteoblasts cultured with Tβms alone showed a higher proliferative activity (Figure 3a and Figure S3a). Tβms did not have the sequence similarity to the proliferation-associated factors, such as insulin growth factor, epidermal growth factor, fibroblast growth factor, even mitogen activated protein kinase (MAPK) and MAPK-like proteins, through searching the NCBI protein database and blasting. How the Tβms promoted osteoblast proliferation, it was unknown. On the sixth day, the lower proliferative activity and more active TGF-β1 production in Tβm groups (Figure 3c and S3c) were observed. Osteoblasts have been reported to downregulate the proliferation during the differentiation period, along with downregulated expression of proliferation associated genes and proteins 38. In the study of Bhaskaran et al. 39, they also found that the expression of TGF-β1 and its downstream components were lower during the proliferation phase, but increased in the differentiation phase to facilitate the cell cycle exit. Hence, the slowdown of the cell proliferative activity was related to the cell differentiation state. During this period TGF-β1 probably played some role. ALP activity assay showed little difference between the groups with or without Tβms. The small differences in the active TGF-β1 concentration (< 40 pg/mL) may be not enough to cause the change of ALP activity. In the study of Noda et al.40, TGF-β1 upregulated the ALP activity in osteoblastic cells in a dose-dependent manner with an ED50 about 0.2 ng/mL. In the presence of 2 ng/mL exogenous TGF-β1, the effect of Tβms as a pro-proliferative agent on osteoblast was impaired in the first four days (Figure 4b and Figure S4a). However, TGF-β1 alone did not induce a repression effect on the osteoblast proliferation (0-4days). In some studies, TGF-β1 inhibited the osteoblast growth when the concentration was above 0.5 ng/mL 40-42. However, under the same range of concentration the promotion of TGF-β1 on osteoblast proliferation was also

reported

43

. Difference in culture

conditions, such as TGF-β1 concentration, cell density, cell differentiation stage, duration 16 ACS Paragon Plus Environment

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of stimulation, the presence of serum or other growth factors, always lead to diverse or even opposite effects of TGF-β1 on osteoblast proliferation and differentiation

34, 44

.

RT-PCR results revealed that the addition of TGF-β1 upregulated the expression of ALP and COL I, but downregulated the expression of Runx2, OPN, OCN and TGF-β1 (Figure 4d). However, no significant difference in the TβRII expression and protein level of p-Smad2/3 was found between the blank control and the TGF-β1 group at 6 d. It seemed that the change of cytokine milieu led to a distinct downstream response upon phosphorylation of Smad2/3. Runx2 is a major target of TGF-β1. It mediates some effects of TGF-β1 on osteoblast differentiation. The promoters of ALP, COL I, OPN and OCN all contain Runx2 binding sites (osteoblast specific element 2, OSE2)

45

. Moreover, Runx2

can also bind to the OSE2 element in its own promoter 19. It means that the repression of Runx2 function will inhibit both the expression of Runx2 and the Runx2-dependent genes. Whether a target gene is to be activated or repressed by TGF-β1, it is further defined by a set of transcriptional coactivators and corepressors

46-48

. They are recruited and interact

with the Smad transcriptional complexes to modulate the transcription of target genes. Researchers have found that TGF-β1 (1-5 ng/mL) inhibits Runx2 function through recruitment of HDACs by Smad3

18-19

. The matrix mineralization, expression of Runx2

and osteogenic differentiation markers, such as ALP and OCN, were all repressed. In this work, RT-PCR analysis showed that compared with the blank control, the decreased expression of OPN and OCN by TGF-β1 was roughly corresponded to the decrease in Runx2 level (Figure 4d-e and Figure S4c-d). However, TGF-β1-mediated repression on Runx2 transcription did not downregulated the ALP and COL I expression on both day 6 and 10. Together with the observation that Tβm4 could block the immunoreactivity of TGF-β1, and osteoblasts in the T+Tβm4 group significantly reduced the p-Smad2/3 protein level, ALP, COL I and TβRII mRNA levels, but increased OCN level, it suggested that 17 ACS Paragon Plus Environment


exogenous TGF-β1-Smad signaling promoted the ALP and COL I expression, but repressed the Runx2, OPN and OCN expression. This deduction was more confirmed by the results that the T+Tβm14 and T+Tβm16 groups had a higher expression of ALP, COL I and TβRII, higher protein level of p-Smad2/3, while lower expression of OCN than the T+ Tβm4 group, due to the greater immunoreactivity of TGF-β1 kept by Tβm14 and Tβm16 (Figure 4a). In addition, it was observed that TGF-β1 expression (autocrine TGF-β1) in the T+Tβm14 and T+Tβm16 groups was upregulated at 10 d (Figure S4d). Compared to the TGF-β1 group, TβmII and OPN levels and phosphorylation of Smad2/3 in these two groups were all upregulated. The T+Tβm16 group also presented an increased Runx2 level. These data led us to a hypothesis that autocrine and exogenous TGF-β1 induced different levels of target gene expression through regulation of Runx2 expression and its transcription function. It was noted that although the blank control always kept the high TGF-β1 expression (Figure 4d-e) and phosphorylation of Smad2/3 (Figure 5), it still showed relatively high expression of those osteogenic differentiation genes. In the opinion of Alliston et al.

19

, autocrine TGF-β signaling exerts both positive and negative role in

osteogenic differentiation. Change of TGF-β concentration gradient and intervention of other signaling pathways are crucial to the switch of TGF-β1 role as an inducer or an inhibitor of osteoblastic differentiation 8, 15, 26, 49-51. In the report of Suzuki et al. 15, the state of Akt phosphorylation determined the TGF-β1 effects on MC3T3-E1 cell differentiation. In this work, addition of exogenous TGF-β1 may make an effect on the function of autocrine TGF-β1. In terms of changes in gene expression from 6 th day to 10 th day (Figure S5), the blank control enormously downregulated the Runx2 expression. Despite the initiation of major bone matrix gene expression during the early stages of osteoblast differentiation, Runx2 is not essential for the maintenance of these gene expressions 36. Its 18 ACS Paragon Plus Environment

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expression level is low in mature osteoblasts, or it will inhibit osteoblast maturation and cause osteopenia with multiple fractures

52

. Therefore, the downregulation of Runx2

predicted the cell progression into mature stage. Although the osteoblasts in the TGF-β1 group was dedicated to the OCN expression after culturing for 6 days (Figure S5), the OCN expression level was still far lower than that in the blank control (Figure 3e). It was suggested that the exogenous TGF-β1 slowed down the osteoblast mature with the inhibitory effect on OCN acting at least in part at the transcriptional level. In terms of Ca deposition (Figure 4f and Figure S4e), 2 ng/mL TGF-β1 was still effective in improving mineralization in vitro culture. This result was consistent with the study of Lieb et al.

53

about the effect of different doses of TGF-β1 (1-20 ng/mL) on bonelike tissue formation. Promotion on early osteoblast differentiation (such as ALP expression) by TGF-β1 may contribute to the improvement of Ca mineralization. Equally, the relatively lower Ca deposition in the T+Tβm4 group may own to the lower ALP activity and expression in the early days.

In addition, one point needed to mention is that OPN is an inhibitor of apatite

crystal growth

37, 54

. Instead of being progressively downregulated towards the end of the

culture, OPN expression was significantly upregulated by T+Tβm4 and T+Tβm14 from 6 th day to 10 th day (Figure S5). Taken together, Tβm16 with greater affinity to TGF-β1 maintained a greater TGF-β1 immunereactivity. In osteoblast culture with TGF-β1, it made a moderate effect on TGF-β1 function, resulting in the best bone mineralization.

CONCLUSION Affinity peptides of TGF-β1 were identified from the phage display library, and they had some sequence similarity to the ligand binding traps, such as LAP and decorin. The selected Tβms displayed the capacity to weaken TGF-β1 signaling through decreasing the phosphorylation of Smad2/3. Tβms individually enhanced osteoblast early proliferation. In 19 ACS Paragon Plus Environment


Page 20 of 32

synergy with exogenous TGF-β1 , they raised the OCN expression, so as to decrease the suppression of TGF-β1 on progression of osteoblast differentiation. Compared to the blank control, Tβms with TGF-β1 improved the ALP activity and Ca deposition. Among the Tβm4, Tβm14 and Tβm16, Tβm4 exhibited a strong similarity to the decorin. As expected, it showed a stronger blockage on the TGF-β1 immunereactivity, and subsequent TGF-β1-receptors response. By contrast, Tβm16 with greater affinity to TGF-β1 maintained a greater TGF-β1 immunereactivity. Its moderate effect on osteoblast proliferation and differentiation may be more favorable for osteogenesis. As mentioned earlier, TGF-β1 plays an important role in a large number of biological processes. In the TGF-β1 null mice, the impaired bone maturation, in addition directly to TGF-β1 deficiency, was strongly impacted by the increased expression of inflammatory cytokines

1, 12

. Hence,

inflammatory response of these peptides are being investigated to synthetically evaluate their roles in osteogenesis.

Supporting Information Analysis of amino acid distribution of Tβms, images of agar plates in the plaque reduction test, cell viability assay, ALP activity assay and active TGF-β1 assay in the groups of Tβm2, Tβm4, Tβm9 and Tβm14, cell viability, ALP activity assay and RT-PCR analysis in the groups of T+Tβm4 and T+Tβm14, relative gene expression on day 10 normalized to day 6, alizarin red staining of osteoblast mineralization, results of screening phage display library, similarity analysis of Tβm sequences with the TGF-β related molecules, comparition of Tβm2, Tβm4, Tβm9, Tβm14 and Tβm16 in detail, RT-PCR primers.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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Acknowledgments This work was supported by the National Natural Science Foundation of China (31570955), National Key Research and Development Program of China (2017YFB0702602) and Applied Basic Research Programs of Sichuan Province, China (2015JY0036).

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45. Harada, H.; Tagashira, S.; Fujiwara, M.; Ogawa, S.; Katsumata, T.; Yamaguchi, A.; Komori, T.; Nakatsuka, M., Cbfa1 Isoforms Exert Functional Differences in Osteoblast Differentiation. The Journal of biological chemistry 1999, 274 (11), 6972–6978. 46. Massagué, J.; Seoane, J.; Wotton, D., Smad transcription factors. GENES & DEVELOPMENT 2005, 19 (23), 2783-810. DOI: 10.1101/. 47. Gaarenstroom, T.; Hill, C. S., TGF-beta signaling to chromatin: how Smads regulate transcription during self-renewal and differentiation. Seminars in cell & developmental biology 2014, 32, 107-18. DOI: 10.1016/j.semcdb.2014.01.009. 48. Jenster, G., Coactivators and corepressors as mediators of nuclear receptor function: An update. Molecular and Cellular Endocrinology 1998, 143, 1–7. 49. Hynes, R. O., The extracellular matrix: not just pretty fibrils. Science 2009, 326 (5957), 1216-9. DOI: 10.1126/science.1176009. 50. Manzano-Moreno, F. J.; Medina-Huertas, R.; Ramos-Torrecillas, J.; Garcia-Martinez, O.; Ruiz, C., The effect of low-level diode laser therapy on early differentiation of osteoblast via BMP-2/TGF-beta1 and its receptors. Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-Maxillo-Facial Surgery 2015, 43 (9), 1926-32. DOI: 10.1016/j.jcms.2015.08.026. 51. Yokota, J.; Chosa, N.; Sawada, S.; Okubo, N.; Takahashi, N.; Hasegawa, T.; Kondo, H.; Ishisaki, A., PDGF-induced PI3K-mediated signaling enhances the TGF-beta-induced osteogenic differentiation of human mesenchymal stem cells in a TGF-beta-activated MEK-dependent manner. International journal of molecular medicine 2014, 33 (3), 534-42. DOI: 10.3892/ijmm.2013.1606. 52. Liu, W.; Toyosawa, S.; Furuichi, T.; Kanatani, N.; Yoshida, C.; Liu, Y.; Himeno, M.; Narai, S.; Yamaguchi, A.; Komori, T., Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. The Journal of cell biology 2001, 155 (1), 157-66. DOI: 10.1083/jcb.200105052. 53. Lieb, E.; Milz, S.; Vogel, T.; Hacker, M.; Dauner, M.; Schulz, M. B., Effects of transforming growth factor beta1 on bonelike tissue formation in three-dimensional cell culture. I. Culture conditions and tissue formation. Tissue engineering 2004, 10 (9-10), 1399-1413. DOI: 10.1089/ten.2004.10.1399. 54. Hunter, G. K.; Hauschka, P. V.; Poole, R. A.; Rosenberg, L. C.; Goldberg, H. A., Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochemical Journal 1996, 317 (1), 59-64. DOI: 10.1042/bj3170059.



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TABLES Table 1: Analysis of Tβm sequences

Tβm

Sequence of peptides*

Molecular weight

Theoretical pI

Charge numbers

Gravy

Repeat times

9.76 5.05 8.76 6.74 9.99 4.35 9.99 3.42 5.98 9.4 8.59 8.22 8.75 6.96 3.42 6.46 8.6

+3 0 +2 +1 +3 -1 +3 -3 +1 +2 +1 +1 +1 +2 -3 +1 +2

-0.433 -1.358 0.058 -0.950 -0.483 0.225 -0.042 0.267 -1.225 -1.508 -0.683 -0.700 -1.050 -1.575 -0.608 -0.717 -0.675

5 2 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1

HTSSLWHLFRST 1471.64D 1 HPHDYNDLTSPF 1442.51D 2 HVTLRYMHPMVS 1470.77D 3 FHNTPYTPPVTN 1387.51D 4 HYKPSLRASALW 1428.66D 5 AHGFYWDEILPV 1446.63D 6 HSKAFPVLYPLR 1427.71D 7 LSLADDPALTDA 1201.30D 8 HLTTTHPEPPYG 1349.47D 9 AFDRYYYPTKTK 1552.75D 10 FPWYKWRLPDVS 1593.85D 11 GAMAQNPDCIRK 1303.52D 12 IPLGRDGGSYQR 1318.45D 13 ADWYHWRSHSSS 1518.57D 14 SDQVADSSNFDL 1297.3D 15 SGVYKVAYDWQH 1452.59D 16 HLSLPLWKWEKS 1523.80D 17 Gravy: Grand average of hydropathicity.

Table 2: Results of phage reduction assay. rhTGF-β1 concentrations of 20 ug/mL and 5ug/mL were used. Phage clone Phage counts (×109)

Phage reduction

TGF-β1 concentration

P-Tβm2

P-Tβm4

P-Tβm9

P-Tβm14

P-Tβm16

0

2700

1620

3060

3360

4200

5µg/mL

2256

1490

1539

1456

1256

20µg/mL

941

228

325

171

221

5µg/mL

16.44%

8.02%

49.71%

56.67%

70.10%

20µg/mL

65.15%

85.93%

89.38%

94.91%

94.74%

The value of phage reduction was determined as: [(pfu no rhTGF-β1−pfu rhTGF-β1 added) / ( pfu no rhTGF-β1)] ×100%.


Page 27 of 32

FIGURES

*

0.6 Amount of TGF-β 1 (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


* *

0.5

*

0.4 0.3 0.2 0.1 0.0

Blank Tβm2

Tβm4

Tβm9

Tβm14

Tβm16

Figure 1: ELISA assay of the binding of 1 ng/mL TGF-β1 on Tβm-coated plates. The binding efficiency was about 38%, 29%, 30%, 41% and 45%, respectively. Data represent the mean ± SEM from 3 experiments (n = 4). *p < 0.05.



a)

b)

c)

d)

e)

Figure 2: Prediction structures of (a) Tβm2; (b) Tβm4; (c) Tβm9; (d) Tβm14 and (e)Tβm16. The left images were prediction results of secondary structures by DNASTAR software using Gamier-Robson method. The right images were molecular simulation results by Hyperchem 7.5 software using the CHARMM 27 force field. The backbone was yellow.


Page 28 of 32

1.6 1.4

Blank 10ug/mL 20ug/mL 50ug/mL

1.2

Tβm16

*

1.0

*

* *

0.8 0.6 0.4

* *

0.2 0.0

2d

4d

6d

b) 0.20

0.15

Blank 10ug/mL 20ug/mL 50ug/mL

*

Tβm16

*

0.10

0.05

0.00

c) Active TGF-β 1 in supernatant (ng/mL)

a)

OD Value

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ALP activity (U/mgprot)

Page 29 of 32

0.18 0.15 0.12

6d

* *

0.09 0.06 0.03 0.00

4d

Tβm16 Blank 10ug/mL 20ug/mL 50ug/mL

6d

Figure 3: Effect of Tβm16 on osteoblast proliferation and differentiation. Different concentrations were investigated. a) cell viability assay at 2 d, 4 d and 6 d; b) ALP activity assay at 4 d and 6 d; c) active TGF-β1 assay in the supernatant at 6 d. Data represent the mean ± SEM from 3 separate experiments (n = 4). *p < 0.05.



* * 36.8%28.1% OD Value

0.4

0.3

0.2

0.2 0.1

0.0

Blank Tβm4 Tβm14 Tβm16 Tβm4 Tβm14 Tβm16

_

_

_

+

+

0.20

0.0

+

2d

d) 60

BlanK TGF-β 1 T+Tβ m16

0.25

30

* *

0.10

0.05

*

3

*

2

*

*

* *

* **

0

Runx2

6d

10d

Blank TGF-β1 T+Tβm16

*

* 4 3

*

*

**

*

*

*

0

Runx2

ALP

COL I

OPN

OCN

ALP

COL I

f) Ca concentration (mM/mgprot)

4d

1


4

1

0.00

2

6d

*

*

15

* *

4d

6d

45

0.15

8

*

*

0.4

39.7%

0.6

c)

e)


0.5

0.8

1 ng/mL TGF-β1 +

ALP activity (U/mgprot)

b)

*

1.0

Fold change

TGF-β 1 immunoreactivity with or without Tβ ms

a)

Fold change

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 32

0.8

*

0.7

OPN

OCN

TGFβ1 Tgfbr2 Tgfb1 TβRII

BlanK TGF-β1 T+Tβm16

0.6

* *

0.5 0.4 0.3 0.2 0.1 0.0

TGFβ1 Tgfbr2 TβRII Tgfb1

6d

10d

Figure 4: Effect of TGF-β1 and Tβms on osteoblast proliferation and differentiation: a) ELISA assay of the blocking efficiency of TGF-β1 immunoreactivity by Tβm4, Tβm14 and Tβm16. Data reflect the mean ± SEM of quadruplicate wells. The blocking efficiency was 39.7%, 36.8% and 28.1%, respectively. b) Cell viability assay at 2 d, 4 d and 6 d. Data represent the mean ± SEM (n = 4); c) ALP activity assay at 4 d and 6 d. Data represent the mean ± SEM (n = 4); d) and e) RT-PCR analysis at 6 d and 10 d, respectively. Cycle threshold (Ct) values were normalized to β-actin expression and values in TGF-β1 group for each gene were used to calculate fold change. Data are representative of 3 separate experiments (n = 3); f) Ca deposition assay at 6 d and 10 d. Data represent the mean ± SEM (n = 4). *p < 0.05.


Page 31 of 32

a)

1.0

0.8

10 d

6d

* *

*

0.5

*

Ratio of p-Smad2/3 / GAPDH

b) Ratio of p-Smad2/3 / GAPDH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6

0.4

0.2

0.0

*

0.4

*

*

0.3

*

0.2

0.1

0.0 blank TGFTβ1 T+TβTm4 T+TβTm14 T+TTβm16 Blank

blank TGF-Tβ1 T+TβT Blank m4 T+TβTm14 T+TTβm16

Figure 5: a) Western bolt analysis of p-Smad2/3 at 6 d and 10 d with GAPDH as a reference (Data are representative of 3 separate experiments); b) Quantitation (mean ± SEM) of pSmad2/3 with ImageJ software from 3 experiments. *p < 0.05.



TABLE OF CONTENTS (TOC) GRAPHIC


Page 32 of 32

Regulation of Osteoblast Differentiation by Affinity Peptides of TGF-Î²1

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