Co-Culturing of Osteoblasts and Chondrocytes Upregulates HIF-1 Pathway of Chondrocytes via MAPK Signaling

LI Jia-chi, ZHANG De-mao, XIE Jing, ZHOU Xue-dong

Abstract

To study the effect of co-culturing chondrocytes with osteoblasts on hypoxia-inducible factor (HIF)-1 pathway of chondrocytes and its mechanism.   Methods  Chondrocytes and osteoblasts were separately extracted from the knee joint and skull of newborn mice by trypsin digestion. The co-culturing system of osteoblasts and chondrocytes was constructed by using Transwell inserts to culture the osteoblasts and 6-well plate to culture the chondrocytes. We used qRT-PCR to examine changes in the mRNA expression of HIFs and its target gene pyruvate dehydrogenase kinase 1 (PDK1) in chondrocytes co-cultured for 24 h. Western blot was used to analyze changes in the protein expression of HIFs and PDK1 and the changes in the activation of mitogen activated protein kinase (MAPK) signaling pathway after the cells were co-cultured for 48 h. Reactive oxygen species (ROS) staining was done to show the changes of ROS production in chondrocytes co-cultured for 48 h.   Results  The results of qRT-PCR and Western blot showed upregulated levels of HIF-1α gene and protein expression (P<0.05) in the chondrocytes after they were co-cultured with osteoblasts. The gene and protein expression levels of PDK1, the target gene of HIF-1, were also upregulated (P<0.05). ROS staining showed that co-culturing of chondrocytes with osteoblasts decreased ROS production in chondrocytes. Western blot revealed that extracellular signal-regulated kinase (ERK) 1/2 and p38 signaling of co-cultured chondrocytes were enhanced (P<0.05).   Conclusion  Co-culturing with osteoblasts enhanced the ERK1/2 and p38 signaling of chondrocytes and upregulated the HIF-1 pathway of chondrocytes.

 

Keywords: Co-culture, Chondrocyte, HIF-1α, ROS

 

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References


GU X X， TANG Z Z， HE Y L， et al. A functional polymorphism in HIF-3α is related to an increased risk of ischemic stroke. J Mol Neurosci， 2021，71(5): 1061–1069.

SEMBA H， TAKEDA N， ISAGAWA T， et al. HIF-1α-PDK1 axis-induced active glycolysis plays an essential role in macrophage migratory capacity. Nat Commun， 2016， 7: 11635[2021-10-09]. https://www. nature.com/articles/ncomms11635. doi: 10.1038/ncomms11635.

JAKUBCZYK K， DEC K， KAŁDUŃSKA J， et al. Reactive oxygen species—Sources, functions, oxidative damage. Pol Merkur Lekarski， 2020，48(284): 124–127.

MOLONEY J N， COTTER T G. ROS signalling in the biology of cancer. Semin Cell Dev Biol，2018，80: 50–64.

YANG S， LIAN G. ROS and diseases: Role in metabolism and energy supply. Mol Cell Biochem，2020，467(1/2): 1–12.

FINDLAY D M， ATKINS G J. Osteoblast-chondrocyte interactions in osteoarthritis. Curr Osteoporos Rep，2014，12(1): 127–134.

NAKAOKA R， HSIONG S X， MOONEY D J. Regulation of chondrocyte differentiation level via co-culture with osteoblasts. Tissue Eng，2006， 12(9): 2425–2433.

JIANG J， NICOLL S B， LU H H. Co-culture of osteoblasts and chondrocytes modulates cellular differentiation in vitro. Biochem Biophys Res Commun，2005，338(2): 762–770.

PENG F， WANG J H， FAN W J， et al. Glycolysis gatekeeper PDK1 reprograms breast cancer stem cells under hypoxia. Oncogene，2018， 37(8): 1062–1074.

XU W N， ZHENG H L， YANG R Z， et al. HIF-1α regulates glucocorticoid-induced osteoporosis through PDK1/AKT/mTOR signaling pathway. Front Endocrinol (Lausanne)， 2019， 10: 922[2021-10-09]. https://doi.org/10.3389/fendo.2019.00922.

MASOUD G N， LI W. HIF-1α pathway: Role, regulation and intervention for cancer therapy. Acta Pharm Sin B，2015，5(5): 378–389.

KARAGIOTA A， KOURTI M， SIMOS G， et al. HIF-1α-derived cell-penetrating peptides inhibit ERK-dependent activation of HIF-1 and trigger apoptosis of cancer cells under hypoxia. Cell Mol Life Sci，2019， 76(4): 809–825.

FREDE S， STOCKMANN C， FREITAG P， et al. Bacterial lipopolysaccharide induces HIF-1 activation in human monocytes via p44/42 MAPK and NF-κB. Biochem J，2006，396(3): 517–527.

YAN L， CAO X， ZENG S， et al. Associations of proteins relevant to MAPK signaling pathway (p38MAPK-1, HIF-1 and HO-1) with coronary lesion characteristics and prognosis of peri-menopausal women. Lipids Health Dis，2016，15(1): 187.

GAO W， HE R， REN J， et al. Exosomal HMGB1 derived from hypoxia-conditioned bone marrow mesenchymal stem cells increases angiogenesis via the JNK/HIF-1α pathway. FEBS Open Bio，2021，11(5): 1364–1373.

EMERLING B M， PLATANIAS L C， BLACK E， et al. Mitochondrial reactive oxygen species activation of p38 mitogen-activated protein kinase is required for hypoxia signaling. Mol Cell Biol，2005，25(12): 4853–4862.

KHANDRIKA L， LIEBERMAN R， KOUL S， et al. Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1α levels contribute to emergence of an aggressive phenotype in prostate cancer. Oncogene，2009，28(9): 1248–1260.


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