Sains Malaysiana 52(6)(2023): 1771-1783

http://doi.org/10.17576/jsm-2023-5206-13

Protein Rembesan Sel Stem Pulpa Gigi Susu Manusia (SHED) semasa Pembezaan kepada Sel Osteoblas

(Proteins Secretion of Human Exfoliated Deciduous Teeth Stem Cells (SHED) during Osteoblast Cell Differentiation)

SHAHRUL HISHAM ZAINAL ARIFFIN^1*, ROHAYA MEGAT ABDUL WAHAB², THANALETCHUMI MANOGARAN¹, FARINAWATI YAZID², SITI NUR ZAHIDAH ZAHARI¹, RUS DINA RUS DIN³ & INTAN ZARINA ZAINOL ABIDIN⁴

¹Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

²Centre of Family Dental Health, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan, Malaysia

³Forensic Science Programme, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

⁴Centre for Research and Graduate Studies, University of Cyberjaya, 63000 Cyberjaya, Selangor, Malaysia

Received: 26 January 2023/Accepted: 13 June 2023

Abstrak

Analisis protein rembesan adalah penting untuk mengenal pasti penglibatan protein di dalam sistem biologi seperti mengenal pasti fungsi dan interaksi protein serta tapak jalan yang terlibat semasa pembezaan sel stem. Kajian ini dilakukan untuk mengenal pasti protein rembesan dan tapak jalan dalam pembezaan osteoblas melalui perubahan keamatan pengekspresan protein rembesan hasil aruhan asid askorbik terhadap sel stem pulpa gigi susu (SHED). SHED dirawat dengan 10 µg/mL asid askorbik selama 7 dan 21 hari dan pengekpresan gen ditentukan melalui transkripsi berbalik-tindak balas rantaian polimerase (RT-PCR) menunjukkan pengekspresanGAPDH (gen endogenous dan kawalan), diikuti dengan pengekspresan gen COL1 dan BSP (penanda osteoblas). SHED (hari ke 7 dan 21) dieram dalam medium tanpa serum selama 12 jam sebelum medium dianalisis menggunakan kromatografi cecair-spektrometri jisim/spektrometri jisim (LC-MS/MS). Protein yang sama diekspreskan dalam kawalan (positif dan negatif) serta asid askorbik seterusnya ditentukan proses biologi dan sekretom berkaitan osteoblas serta mempunyai perubahan keamatan pengekspresan melalui UniProt dan PANTHER. Hubung kait antara protein-protein rembesan ini bagi mengenal pasti tapak jalan pembezaan osteoblas ditentukan melalui STRING. Sebanyak 57 protein yang sama diekspreskan semasa aruhan asid askorbik dan kawalan dengan empat protein rembesan berkaitan dengan osteoblas didapati mengalami perubahan keamatan pengekspresan. Bagi hubung kait antara protein pula hanya tiga dari empat protein rembesan iaitu gelsolin (GSN), kolagenase jenis IV 72 kDa (MMP2) dan homolog peroksidasin (PXDN) dikenal pasti terlibat dalam tiga tapak jalan. Kesimpulannya, tiga jenis protein rembesan yang melibatkan tiga tapak jalan berpotensi sebagai penanda protein rembesan semasa pembezaan SHED kepada osteoblas.

Kata kunci: Asid askorbik; pembezaan osteoblas; protein rembesan; sel stem pulpa gigi susu manusia (SHED)

Abstract

Analysis of secreted proteins is crucial in understanding proteins’ involvement in the biological system, namely the function and interactions of the proteins and pathways involved, especially during stem cell differentiation. This study is aimed to determine the secreted proteins and pathways during osteoblast differentiation of ascorbic acid-induced exfoliated deciduous teeth stem cells (SHED) via changes in the intensity of secreted proteins’ expression results. SHED was treated with 10 µg/mL ascorbic acid for 7 and 21 days, and genes expression was determined using reverse transcriptase-polymerase chain reaction (RT-PCR), which resulted in the expressed of GAPDH (endogenous gene and control), followed by the expression of COL1 and BSP genes (osteoblast marker). SHEDS (at days 7 and 21) were incubated in a serum-free basal medium for 12 h before being analysed using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Based on similar proteins expressed in controls (positive and negative) and ascorbic acid; the biological processes and osteoblast-related secreted proteins that experienced changes in expression intensity were determined using UniProt and PANTHER. The interaction between secreted proteins was determined using STRING to establish pathways involved during SHED-osteoblast differentiation. A total of 57 similar proteins were identified in ascorbic acid induction and controls of which only four secreted proteins were discovered to be associated with osteoblast and experiencing changes in expression intensity. Regarding protein interaction, only three out of four proteins; gelsolin (GSN), 72 kDa type IV collagenase (MMP2), and peroxidasin homolog (PXDN) were involved in three pathways. In conclusion, the three types of secreted proteins involved in three pathways are suggested as secreted proteins markers during osteoblast differentiation of SHED.

Keywords: Ascorbic acid; osteoblast differentiation; protein secretion; stem cells from human deciduous tooth pulp (SHED)

REFERENCES

Baker, J.A., Wong, W.C., Eisenhaber, B., Warwicker, J. & Eisenhaber, F. 2017. Charged residues next to transmembrane regions revisited: "positive-inside rule" is complemented by the "negative inside depletion/outside enrichment rule". BMC Biol. 15(1): 66. doi:10.1186/s12915-017-0404-4

Brandizzi, F. & Barlowe, C. 2013. Organization of the Er-Golgi interface for membrane traffic control. Nat. Rev. Mol. Cell. Biol. 14(6): 382-392. doi:10.1038/nrm3588

Chang, H.Y., Hor, S.Y., Lim, K.P., Zain, R.B., Cheong, S.C., Rahman, M.A. & Karsani, S.A. 2013. Oral cancer secretome: Identification of cancer-associated proteins. Electrophoresis 34(15): 2199-2208. doi:10.1002/elps.201300126

Chen, Y.Y., He, S.T., Yan, F.H., Zhou, P.F., Luo, K., Zhang, Y.D., Xiao, Y. & Lin, M.K. 2016. Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue. International Journal of Oral Sciences 8(4): 213-222. doi:10.1038/ijos.2016.33

Chevallet, M., Diemer, H., Van Dorssealer, A., Villiers, C. & Rabilloud, T. 2007. Toward a better analysis of secreted proteins: The example of the myeloid cells secretome. Proteomics 7(11): 1757-1770. doi:10.1002/pmic.200601024

Choi, Y.A., Lim, J., Kim, K.M., Acharya, B., Cho, J.Y., Bae, Y.C., Shin, H.I., Kim, S.Y. & Park, E.K. 2010. Secretome analysis of human BMSCs and identification of SMOC1 as an important ecm protein in osteoblast differentiation. J. Proteome. Res. 9(6): 2946-2956. doi:10.1021/pr901110q

Cypher, T.J. & Grossman, J.P. 1996. Biological principles of bone graft healing. J. Foot Ankle Surg. 35(5): 413-417. doi: 10.1016/s1067-2516(96)80061-5

Dalmer, T.R.A. & Clugston, R.D. 2019. Gene ontology enrichment analysis of congenital diaphragmatic hernia-associated genes. Pediatr. Res. 85(1): 13-19. doi:10.1038/s41390-018-0192-8

Deng, C., He, Y., Feng, J., Dong, Z., Yao, Y., Mok, H., Lin, M. & Feng, L. 2017. Extracellular matrix/stromal vascular fraction gel conditioned medium accelerates wound healing in a murine model. Wound Repair Regen. 25(6): 923-932. doi:10.1111/wrr.12602

Deracinois, B., Flahaut, C., Duban-Deweer, S. & Karamanos, Y. 2013. Comparative and quantitative global proteomics approaches: An overview. Proteomes 1(3): 180-218. doi:10.3390/proteomes1030180

Dou, L., Wu, Y., Yan, Q., Wang, J., Zhang, Y. & Ji, P. 2017. Secretome profiles of immortalized dental follicle cells using itraq-based proteomic analysis. Scientific Reports 7(1): 1-8. doi:10.1038/s41598-017-07467-3

Farinawati, Y., Ng, W.C., Nur Atmaliya, L., Shahrul Hisham, Z.A. & Rohaya, M.A.W. 2022. Higher concentration of ascorbic acid as a sole induction factor for osteogenesis on MC3T3-E1 cell model. Sains Malaysiana 51(5): 1449-1464. doi:10.17576/jsm-2022-5105-15

Farshdousti Hagh, M., Noruzinia, M., Mortazavi, Y., Soleimani, M., Kaviani, S., Abroun, S., Dehghani Fard, A. & Mahmoodinia, M. 2015. Different methylation patterns of RUNX2, OSX, DLX5 and BSP in osteoblastic differentiation of mesenchymal stem cells. Cell J. 17(1): 71-82. doi:10.22074/cellj.2015.513

Gattazzo, F., Urciuolo, A. & Bonaldo, P. 2014. Extracellular matrix: A dynamic microenvironment for stem cell niche. Biochimica et Biophysica Acta (BBA) - General Subjects 1840(8): 2506-2519. doi:10.1016/j.bbagen.2014.01.010

Han, Y., You, X., Xing, W., Zhang, Z. & Zou, W. 2018. Paracrine and endocrine actions of bone-the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts. Bone Res 6(1): 1-11. doi:10.1038/s41413-018-0019-6

Iaquinta, M.R., Mazzoni, E., Bononi, I., Rotondo, J.C., Mazziotta, C., Montesi, M., Sprio, S., Tampieri, A., Tognon, M. & Martini, F. 2019. Adult stem cells for bone regeneration and repair. Frontiers in Cell and Developmental Biology 7: 268. doi:10.3389/fcell.2019.00268

Intan Zarina, Z.A., Manogaran, T., Rohaya, M.A.W., Saiful Anuar, K., Muhammad Dain, Y., Farinawati, Y., Zaidah, Z.A., Anis Nabilah, J. & Shahrul Hisham, Z.A. 2023. Label-free quantitative proteomic analysis of differential expressed osteoblast-related proteins in dental pulp stem cells from deciduous and permanent teeth. Current Stem Cell Research and Therapy 18(3): 417-428. doi:10.2174/1574888X17666220627145424

Intan Zarina, Z.A., Manogaran, T., Rohaya, M.A.W., Farinawati, Y. & Shahrul Hisham, Z.A. 2022. A comparative analysis of ascorbic acid-induced cytotoxicity and differentiation between SHED and DPSC. Current Stem Cell Research and Therapy 17(6): 576-588. doi:10.2174/1574888X17666220124141310

Jafary, F., Hanachi, P. & Gorjipour, K. 2017. Osteoblast differentiation on collagen scaffold with immobilized alkaline phosphatase. International Journal of Organ Transplantation Medicine 8(4): 195-202.

Katagiri, W., Sakaguchi, K., Kawai, T., Wakayama, Y., Osugi, M. & Hibi, H. 2017. A defined mix of cytokines mimics conditioned medium from cultures of bone marrow-derived mesenchymal stem cells and elicits bone regeneration. Cell Prolif 50(3): e12333. doi:10.1111/cpr.12333

Kim, J.M., Kim, J., Kim, Y.H., Kim, K.T., Ryu, S.H., Lee, T.G. & Suh, P.G. 2013. Comparative secretome analysis of human bone marrow-derived mesenchymal stem cells during osteogenesis. J. Cell Physiol. 228(1): 216-224. doi:10.1002/jcp.24123

Kim, J., Gee, H.Y. & Lee, M.G. 2018. Unconventional protein secretion - New insights into the pathogenesis and therapeutic targets of human diseases. Journal of Cell Science 131(12): jcs213686. doi:10.1242/jcs.213686

Koh, B., Nadiah, S., Sharifah Nursyazwani Shahirah, W.I., Roszalina, R., Siti Salmiah, M.Y., Ruszymah, B.H.I., Shahrul Hisham, Z.A., Rohaya, M.A.W. & Muhammad Dain, Y. 2021. Mesenchymal stem cells: A comprehensive methods for odontoblastic induction. Biological Procedure Online 23(1): 1-13. doi:10.1186/s12575-021-00155-7

Kruger, T.E., Miller, A.H. & Wang, J. 2013. Collagen scaffolds in bone sialoprotein-mediated bone regeneration. The Scientific World Journal 2013: Article ID. 812718: 6. doi:10.1155/2013/812718

Launay, G., Ceres, N. & Martin, J. 2017. Non-interacting proteins may resemble interacting proteins: Prevalence and implications. Sci. Rep. 7(1): 40419. doi:10.1038/srep40419

Liang, H.P.H., Xu, J., Xue, M. & Jackson, C.J. 2016. Matrix metalloproteinases in bone development and pathology: Current knowledge and potential clinical utility. Metalloproteinases in Medicine 3: 93-102. doi:10.2147/MNM.S92187

Lopes, T.J.S., Schaefer, M., Shoemaker, J., Matsuoka, Y., Fontaine, J.F., Neumann, G., Andrade-Navarro, M.A., Kawaoka, Y. & Kitano, H. 2011. Tissue-specific subnetworks and characteristics of publicly available human protein interaction databases. Bioinformatics 27(17): 2414-2421. doi:10.1093/bioinformatics/btr414

Madsen, D.H., Jurgensen, H.J., Ingvarsen, S., Melander, M.C., Albrechtsen, R., Hald, A., Holmbeck, K., Bugge, T.H., Behrendt, N. & Engelholm, L.H. 2013. Differential actions of the endocytic collagen receptor uPARAP/Endo180 and the collagenase MMP-2 in bone homeostasis. PLoS ONE 8(8): e71261. doi:10.1371/journal.pone.0071261

Nag, S., Larsson, M., Robinson, R.C. & Burtnick, L.D. 2013. Gelsolin: The tail of a molecular gymnast. Cytoskeleton (Hoboken) 70(7): 360-384. doi:10.1002/cm.21117

Nur Atmaliya, L., Rohaya, M.A.W., Shahrul Hisham, Z.A., Nurrul Shaqinah, N., Seng, F.L. & Farinawati, Y. 2022. Comparison between hydroxyapatite and polycaprolactone in inducing osteogenic differentiation and augmenting maxillary bone regeneration in rats. PeerJ 10: e13356. doi:10.7717/peerj.13356

Olimpio, R.M.C., De Oliveira, M., De Sibio, M.T., Moretto, F.C.F., Deprá, I.C., Mathias, L.S., Gonçalves, B.M., Rodrigues, B.M., Tilli, H.P., Coscrato, V.E., Costa, S.M.B., Mazeto, G., Fernandes, C.J.C., Zambuzzi, W.F., Saraiva, P.P., Maria, D.A. & Nogueira, C.R. 2018. Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells. PLoS ONE 13(4): e0194847. doi:10.1371/journal.pone.0194847

Péterfi, Z., Donkó, A., Orient, A., Sum, A., Prókai, A., Molnár, B., Veréb, Z., Rajnavölgyi, E., Kovács, K.J., Müller, V., Szabó, A.J. & Geiszt, M. 2009. Peroxidasin is secreted and incorporated into the extracellular matrix of myofibroblasts and fibrotic kidney. Am. J. Pathol. 175(2): 725-735. doi:10.2353/ajpath.2009.080693

Rohaya, M.A.W., Nur Akmal, M.R., Sahidan, S., Intan Zarina, Z.A., Zaidah, Z.A. & Shahrul Hisham, Z.A. 2017. Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells. PeerJ 5: e3180. doi:10.7717/peerj.3180

Romanello, M., Piatkowska, E., Antoniali, G., Cesaratto, L., Vascotto, C., Iozzo, R.V., Delneri, D. & Brancia, F.L. 2014. Osteoblastic cell secretome: A novel role for progranulin during risedronate treatment. Bone 58: 81-91. doi:10.1016/j.bone.2013.10.003

Schmidt, J.R., Kliemt, S., Preissler, C., Moeller, S., Von Bergen, M., Hempel, U. & Kalkhof, S. 2016. Osteoblast-released matrix vesicles, regulation of activity and composition by sulfated and non-sulfated glycosaminoglycans. Mol. Cell Proteomics 15(2): 558-572. doi:10.1074/mcp.M115.049718

Shahrul Hisham, Z.A., Manogaran, T., Rohaya, M.A.W., Saiful Anuar, K., Intan Zarina, Z.A., Farinawati, Y., Muhammad Dain, Y. & Zaidah, Z.A. 2022. Profil protein sekretom sel stem pulpa gigi manusia semasa aruhan asid askorbik. Sains Malaysiana 51(7): 2173-2186. doi:10.17576/jsm-2022-5107-19

Shahrul Hisham, Z.A., Thanaletchumi, M., Intan Zarina, Z.A., Zaidah, Z.A. & Rohaya, M.A. W. 2019. Culture and molecular markers characterization of stem cells from human deciduous (SHED) and permanent (DPSC) teeth pulp. Sains Malaysiana 48(9): 1855-1865. doi:10.17576/jsm-2019-4809-06

Shin, J., Rhim, J., Kwon, Y., Choi, S.Y., Shin, S., Ha, C.W. & Lee, C. 2019. Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using boncat and pulsed silac. Sci. Rep. 9(1): 1-2. doi:10.1038/s41598-019-39650-z

Shotorbani, B.B., André, H., Barzegar, A., Zarghami, N., Salehi, R. & Alizadeh, E. 2018. Cell sheet biofabrication by co-administration of mesenchymal stem cells secretome and vitamin C on thermoresponsive polymer. J. Mater. Sci. Mater. Med. 29(11): 170. doi:10.1007/s10856-018-6180-z

Siti Norhaiza, H., Siti Norsaidah, I., Rohaya, M.A.W., Intan Zarina, Z.A., Sahidan, S., Zaidah, Z.A., Mohamad, A.R. & Shahrul Hisham, Z.A. 2014. Ascorbic acid induces osteoblast differentiation of human suspension mononuclear cells. Cytotherapy 16(5): 674-682. doi:10.1016/j.jcyt.2013.07.013

Szklarczyk, D., Gable, A.L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., Simonovic, M., Doncheva, N.T., Morris, J.H., Bork, P., Jensen, L.J. & von Mering, C. 2018. String V11: Protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Research 47(D1): D607-D613. doi:10.1093/nar/gky1131

Thouverey, C., Malinowska, A., Balcerzak, M., Strzelecka-Kiliszek, A., Buchet, R., Dadlez, M. & Pikula, S. 2011. Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells. J. Proteomics 74(7): 1123-1134. doi:10.1016/j.jprot.2011.04.005

Xiao, Z., Camalier, C.E., Nagashima, K., Chan, K.C., Lucas, D.A., De La Cruz, M.J., Gignac, M., Lockett, S., Issaq, H.J., Veenstra, T.D., Conrads, T.P. & Beck, G.R., Jr. 2007. Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts. J. Cell Physiol. 210(2): 325-335. doi:10.1002/jcp.20826

*Corresponding author; email: hisham@ukm.edu.my