TGF-β effects on adipogenesis of 3T3-L1 cells differ in 2D and 3D cell culture conditions.
3D culture
3T3‐L1 cell
TGF‐β isoform
adipogenesis
Journal
FEBS open bio
ISSN: 2211-5463
Titre abrégé: FEBS Open Bio
Pays: England
ID NLM: 101580716
Informations de publication
Date de publication:
08 Oct 2024
08 Oct 2024
Historique:
revised:
04
04
2024
received:
06
02
2024
accepted:
23
08
2024
medline:
9
10
2024
pubmed:
9
10
2024
entrez:
9
10
2024
Statut:
aheadofprint
Résumé
The TGF-β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF-β, TGF-β-1~3. To further elucidate their role, two-dimensionally (2D) and three-dimensionally (3D) cultured 3T3-L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis-related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3-L1 spheroids. In the 2D cultured 3T3-L1 cells, mRNA expression levels of adipogenesis-related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF-β isoforms, with TGF-β2 having the greater effects. Consistent with these results, treatment with TGF-β2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3-L1 cells. However, the inhibitory effect of TGF-β on adipogenesis decreased under 3D spheroid culture conditions and TGF-β isoforms did not affect adipogenesis-induced (a) enlargement and downsizing of 3T3-L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up-regulation of the mRNA expression of adipogenesis-related genes. The findings presented herein suggest that the three TGF-β isoforms have different suppressive effects on adipogenesis-related cellular properties of 2D cultured 3T3-L1 cells and that their effects decrease under 3D spheroid culture conditions.
Identifiants
pubmed: 39380256
doi: 10.1002/2211-5463.13890
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Références
Friedenstein AJ, Petrakova KV, Kurolesova AI and Frolova GP (1968) Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6, 230–247.
Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF, Rudakowa SF, Luriá EA and Ruadkow IA (1974) Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 2, 83–92.
Mushahary D, Spittler A, Kasper C, Weber V and Charwat V (2018) Isolation, cultivation, and characterization of human mesenchymal stem cells. Cytometry A 93, 19–31.
Compte M, Cuesta AM, Sánchez‐Martín D, Alonso‐Camino V, Vicario JL, Sanz L and Alvarez‐Vallina L (2009) Tumor immunotherapy using gene‐modified human mesenchymal stem cells loaded into synthetic extracellular matrix scaffolds. Stem Cells 27, 753–760.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147.
Lin CS, Xin ZC, Deng CH, Ning H, Lin G and Lue TF (2010) Defining adipose tissue‐derived stem cells in tissue and in culture. Histol Histopathol 25, 807–815.
McBeath R, Pirone DM, Nelson CM, Bhadriraju K and Chen CS (2004) Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6, 483–495.
Bowers RR, Kim JW, Otto TC and Lane MD (2006) Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: role of the BMP‐4 gene. Proc Natl Acad Sci USA 103, 13022–13027.
Konieczny SF and Emerson CP Jr (1984) 5‐Azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: evidence for regulatory genes controlling determination. Cell 38, 791–800.
Cristancho AG and Lazar MA (2011) Forming functional fat: a growing understanding of adipocyte differentiation. Nat Rev Mol Cell Biol 12, 722–734.
Rosen ED, Walkey CJ, Puigserver P and Spiegelman BM (2000) Transcriptional regulation of adipogenesis. Genes Dev 14, 1293–1307.
Tontonoz P, Hu E and Spiegelman BM (1994) Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid‐activated transcription factor. Cell 79, 1147–1156.
Tamori Y, Masugi J, Nishino N and Kasuga M (2002) Role of peroxisome proliferator‐activated receptor‐gamma in maintenance of the characteristics of mature 3T3‐L1 adipocytes. Diabetes 51, 2045–2055.
Imai T, Takakuwa R, Marchand S, Dentz E, Bornert JM, Messaddeq N, Wendling O, Mark M, Desvergne B, Wahli W et al. (2004) Peroxisome proliferator‐activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse. Proc Natl Acad Sci USA 101, 4543–4547.
Galic S, Oakhill JS and Steinberg GR (2010) Adipose tissue as an endocrine organ. Mol Cell Endocrinol 316, 129–139.
Centrella M, Horowitz MC, Wozney JM and McCarthy TL (1994) Transforming growth factor‐beta gene family members and bone. Endocr Rev 15, 27–39.
Massagué J, Cheifetz S, Endo T and Nadal‐Ginard B (1986) Type beta transforming growth factor is an inhibitor of myogenic differentiation. Proc Natl Acad Sci USA 83, 8206–8210.
Ignotz RA and Massagué J (1985) Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts. Proc Natl Acad Sci USA 82, 8530–8534.
Choy L, Skillington J and Derynck R (2000) Roles of autocrine TGF‐beta receptor and Smad signaling in adipocyte differentiation. J Cell Biol 149, 667–682.
Sparks RL, Allen BJ and Strauss EE (1992) TGF‐beta blocks early but not late differentiation‐specific gene expression and morphologic differentiation of 3T3 T proadipocytes. J Cell Physiol 150, 568–577.
Clouthier DE, Comerford SA and Hammer RE (1997) Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy‐like syndrome in PEPCK‐TGF‐beta1 transgenic mice. J Clin Invest 100, 2697–2713.
Samad F, Yamamoto K, Pandey M and Loskutoff DJ (1997) Elevated expression of transforming growth factor‐beta in adipose tissue from obese mice. Mol Med 3, 37–48.
Sparks RL, Allen BJ, Zygmunt AI and Strauss EE (1993) Loss of differentiation control in transformed 3T3 T proadipocytes. Cancer Res 53, 1770–1776.
Bortell R, Owen TA, Ignotz R, Stein GS and Stein JL (1994) TGF beta 1 prevents the down‐regulation of type I procollagen, fibronectin, and TGF beta 1 gene expression associated with 3T3‐L1 pre‐adipocyte differentiation. J Cell Biochem 54, 256–263.
Rahimi N, Tremblay E, McAdam L, Roberts A and Elliott B (1998) Autocrine secretion of TGF‐beta 1 and TGF‐beta 2 by pre‐adipocytes and adipocytes: a potent negative regulator of adipocyte differentiation and proliferation of mammary carcinoma cells. In Vitro Cell Dev Biol Anim 34, 412–420.
Morikawa M, Derynck R and Miyazono K (2016) TGF‐β and the TGF‐β family: context‐dependent roles in cell and tissue physiology. Cold Spring Harb Perspect Biol 8, a021873.
Yu L, Border WA, Huang Y and Noble NA (2003) TGF‐beta isoforms in renal fibrogenesis. Kidney Int 64, 844–856.
Shah M, Foreman DM and Ferguson MW (1995) Neutralisation of TGF‐beta 1 and TGF‐beta 2 or exogenous addition of TGF‐beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 108, 985–1002.
Ashcroft GS, Yang X, Glick AB, Weinstein M, Letterio JL, Mizel DE, Anzano M, Greenwell‐Wild T, Wahl SM, Deng C et al. (1999) Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol 1, 260–266.
Amendt C, Mann A, Schirmacher P and Blessing M (2002) Resistance of keratinocytes to TGFbeta‐mediated growth restriction and apoptosis induction accelerates re‐epithelialization in skin wounds. J Cell Sci 115, 2189–2198.
Ferguson MW, Duncan J, Bond J, Bush J, Durani P, So K, Taylor L, Chantrey J, Mason T, James G et al. (2009) Prophylactic administration of avotermin for improvement of skin scarring: three double‐blind, placebo‐controlled, phase I/II studies. Lancet 373, 1264–1274.
Garweg JG, Zandi S, Gerhardt C and Pfister IB (2017) Isoforms of TGF‐β in the aqueous humor of patients with pseudoexfoliation syndrome and a possible association with the long‐term stability of the capsular bag after cataract surgery. Graefes Arch Clin Exp Ophthalmol 255, 1763–1769.
Igarashi N, Honjo M, Asaoka R, Kurano M, Yatomi Y, Igarashi K, Miyata K, Kaburaki T and Aihara M (2021) Aqueous autotaxin and TGF‐βs are promising diagnostic biomarkers for distinguishing open‐angle glaucoma subtypes. Sci Rep 11, 1408.
Watanabe M, Sato T, Tsugeno Y, Higashide M, Furuhashi M and Ohguro H (2023) TGF‐β‐3 induces different Effects from TGF‐β‐1 and ‐2 on cellular metabolism and the spatial properties of the human trabecular meshwork cells. Int J Mol Sci 24, 4181.
Igarashi N, Honjo M, Yamagishi R, Kurano M, Yatomi Y, Igarashi K, Kaburaki T and Aihara M (2021) Crosstalk between transforming growth factor β‐2 and Autotaxin in trabecular meshwork and different subtypes of glaucoma. J Biomed Sci 28, 47.
Ida Y, Hikage F, Itoh K, Ida H and Ohguro H (2020) Prostaglandin F2α agonist‐induced suppression of 3T3‐L1 cell adipogenesis affects spatial formation of extra‐cellular matrix. Sci Rep 10, 7958.
Ida Y, Hikage F, Umetsu A, Ida H and Ohguro H (2020) Omidenepag, a non‐prostanoid EP2 receptor agonist, induces enlargement of the 3D organoid of 3T3‐L1 cells. Sci Rep 10, 16018.
Itoh K, Hikage F, Ida Y and Ohguro H (2020) Prostaglandin F2α agonists negatively modulate the size of 3D organoids from primary human orbital fibroblasts. Invest Ophthalmol Vis Sci 61, 13.
Ida Y, Hikage F and Ohguro H (2021) ROCK inhibitors enhance the production of large lipid‐enriched 3D organoids of 3T3‐L1 cells. Sci Rep 11, 5479.
Kumar A, Ruan M, Clifton K, Syed F, Khosla S and Oursler MJ (2012) TGF‐β mediates suppression of adipogenesis by estradiol through connective tissue growth factor induction. Endocrinology 153, 254–263.
Singh K, Sachan N, Ene T, Dabovic B and Rifkin D (2022) Latent transforming growth factor β binding protein 3 controls adipogenesis. Matrix Biol 112, 155–170.
Oouchi Y, Watanabe M, Ida Y, Ohguro H and Hikage F (2021) Rosiglitasone and ROCK inhibitors modulate Fibrogenetic changes in TGF‐β2 treated human conjunctival fibroblasts (HconF) in different manners. Int J Mol Sci 22, 7335.
Tsugeno Y, Furuhashi M, Sato T, Watanabe M, Umetsu A, Suzuki S, Ida Y, Hikage F and Ohguro H (2022) FGF‐2 enhances fibrogenetic changes in TGF‐β2 treated human conjunctival fibroblasts. Sci Rep 12, 16006.
Tsugeno Y, Sato T, Watanabe M, Higashide M, Furuhashi M, Umetsu A, Suzuki S, Ida Y, Hikage F and Ohguro H (2022) All trans‐retinoic acids facilitate the remodeling of 2D and 3D cultured human conjunctival fibroblasts. Bioengineering (Basel) 9, 463.
Hikage F, Atkins S, Kahana A, Smith TJ and Chun TH (2019) HIF2A‐LOX pathway promotes fibrotic tissue remodeling in thyroid‐associated Orbitopathy. Endocrinology 160, 20–35.
Ota C, Ida Y, Ohguro H and Hikage F (2020) ROCK inhibitors beneficially alter the spatial configuration of TGFβ2‐treated 3D organoids from a human trabecular meshwork (HTM). Sci Rep 10, 20292.
Schefe JH, Lehmann KE, Buschmann IR, Unger T and Funke‐Kaiser H (2006) Quantitative real‐time RT‐PCR data analysis: current concepts and the novel “gene expression's CT difference” formula. J Mol Med (Berl) 84, 901–910.
Ida Y, Sato T, Umetsu A, Watanabe M, Furuhashi M, Hikage F and Ohguro H (2022) Addition of ROCK inhibitors alleviates prostaglandin‐induced inhibition of Adipogenesis in 3T3L‐1 spheroids. Bioengineering (Basel) 9, 702.
Ida Y, Furuhashi M, Watanabe M, Umetsu A, Hikage F and Ohguro H (2021) Prostaglandin F2 and EP2 agonists exert different effects on 3D 3T3‐L1 spheroids during their culture phase. Biomedicine 9, 1821.
Ohguro H, Ida Y, Hikage F, Umetsu A, Ichioka H, Watanabe M and Furuhashi M (2022) STAT3 is the master regulator for the forming of 3D spheroids of 3T3‐L1 Preadipocytes. Cells 11, 300.
Endo K, Sato T, Umetsu A, Watanabe M, Hikage F, Ida Y, Ohguro H and Furuhashi M (2023) 3D culture induction of adipogenic differentiation in 3T3‐L1 preadipocytes exhibits adipocyte‐specific molecular expression patterns and metabolic functions. Heliyon 9, e20713.
Zamani N and Brown CW (2011) Emerging roles for the transforming growth factor‐{beta} superfamily in regulating adiposity and energy expenditure. Endocr Rev 32, 387–403.
Tan CK, Chong HC, Tan EH and Tan NS (2012) Getting ‘Smad’ about obesity and diabetes. Nutr Diabetes 2, e29.
Yadav H, Quijano C, Kamaraju AK, Gavrilova O, Malek R, Chen W, Zerfas P, Zhigang D, Wright EC, Stuelten C et al. (2011) Protection from obesity and diabetes by blockade of TGF‐β/Smad3 signaling. Cell Metab 14, 67–79.
Huh D, Hamilton GA and Ingber DE (2011) From 3D cell culture to organs‐on‐chips. Trends Cell Biol 21, 745–754.
Wang F, Weaver VM, Petersen OW, Larabell CA, Dedhar S, Briand P, Lupu R and Bissell MJ (1998) Reciprocal interactions between beta1‐integrin and epidermal growth factor receptor in three‐dimensional basement membrane breast cultures: a different perspective in epithelial biology. Proc Natl Acad Sci USA 95, 14821–14826.
Hsiao AY, Tung YC, Qu X, Patel LR, Pienta KJ and Takayama S (2012) 384 hanging drop arrays give excellent Z‐factors and allow versatile formation of co‐culture spheroids. Biotechnol Bioeng 109, 1293–1304.
Hsiao AY, Tung YC, Kuo CH, Mosadegh B, Bedenis R, Pienta KJ and Takayama S (2012) Micro‐ring structures stabilize microdroplets to enable long term spheroid culture in 384 hanging drop array plates. Biomed Microdevices 14, 313–323.
Nandy A and Rendina‐Ruedy E (2021) Bone marrow adipocytes – good, bad, or just different? Best Pract Res Clin Endocrinol Metab 35, 101550.
Kim BH, Han S, Lee H, Park CH, Chung YM, Shin K, Lee HG and Ye SK (2015) Metformin enhances the anti‐adipogenic effects of atorvastatin via modulation of STAT3 and TGF‐β/Smad3 signaling. Biochem Biophys Res Commun 456, 173–178.
Zhang K, Guo W, Yang Y and Wu J (2011) JAK2/STAT3 pathway is involved in the early stage of adipogenesis through regulating C/EBPβ transcription. J Cell Biochem 112, 488–497.