Supervisors info:
Αντώνιος Χατζηγεωργίου, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ, Επιβλέπων
Δημήτρης Κλέτσας, Ερευνητής Α', Διευθυντής Ινστιτούτου Βιοεπιστημών και Εφαρμογών, ΕΚΕΦΕ «Δημόκριτος»
Μιχαήλ Κουτσιλιέρης, Ομότιμος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Intervertebral disc is an elastic fibrocartilaginous structure that is located between successive vertebrae and forms the joint of the spine. Each intervertebral disc is composed of the inner nucleus pulposus (NP) and the outer annulus fibrosus (AF) surrounding it. Intervertebral disc cells are responsible for producing and degrading the components of the extracellular matrix, which provides support, strength, and protection to the intervertebral disc structure.
As the intervertebral disc degenerates, changes occur in its composition, structure and biochemistry. The boundaries between the AF and the NP are no longer distinct, extracellular matrix homeostasis is disrupted and the percentage of senescent cells - characterized by a senescence-associated secretory phenotype (SASP) - increases. Intervertebral disc degeneration is an age-related and multifactorial condition. One of its causes is the irreversible loss of estrogens that occurs during menopause in women. The lack of 17β-estradiol results in the accumulation of matrix metalloproteinases (MMPs) in the extracellular matrix of the intervertebral disc, oxidative stress and inflammation. Studies performed in AF and NP cells have shown that exogenous administration of 17β-estradiol alleviates intervertebral disc degeneration. On the other hand, senescent cells, which have been shown to accumulate in the intervertebral disc, also lead to increased expression and activity of MMPs, oxidative stress and inflammation. For these reasons, the aim of the present study was to assess estrogen receptors’ expression profile, focusing on estrogen receptor GPER, in young and senescent intervertebral disc cells, as well as the possible beneficial effect of exogenous 17β-estradiol administration on these cells.
First, protein expression levels of the estrogen receptors ERα, ERβ and GPER were assessed in young AF and NP cells cultured in two types of culture medium, the classic DMEM supplemented with 10% (v/v) FBS and a phenol red-free DMEM supplemented with 10% (v/v) FBS-DCT (Dextran/Charcoal Treated), which is deprived of estrogens. Western analysis did not show any difference in the receptors’ expression levels dependent on the culture medium type. Furthermore, ERα receptor’s expression was not detectable in these cells, whereas ERβ and GPER showed detectable expression levels in both AF and NP cells. As ERβ is well studied, the estrogen receptor GPER was chosen for further investigation, as there is insufficient data on its role in intervertebral disc degeneration.
Subsequently, cellular senescence was induced in AF cells following exposure to γ-irradiation, as confirmed by the lower BrdU incorporation, the increased expression levels of the biomarkers p16INK4α and p21WAF1, and the positive senescence-specific SA-β-Gal staining. Expression levels of GPER and selected genes regulating tissue homeostasis at the mRNA level were then examined in young and senescent bovine AF cells. RT-qPCR analysis revealed that there was no change in GPER expression between young and senescent AF cells, whereas genes encoding MMP-3, -9 and interleukin-6 were upregulated in senescent AF cells. At the protein level, GPER expression also showed no alteration between young and senescent bovine cells, a finding confirmed in human cells, as well.
The same cycle of experiments was performed in NP cells. Senescent cells were characterized based on the above mentioned criteria and the expression pattern of GPER and selected SASP genes was investigated. A tendency for GPER levels to decrease, an up-regulation of the genes encoding MMP-3 and -9 and HO-1 and a decrease of the levels of the gene encoding type II collagen was found in senescent NP cells. In addition, the decrease in GPER levels in senescent cells was confirmed at the protein level in both bovine and human cells.
Then, to study the effect of 17β-estradiol on intervertebral disc cells, the MTT assay was performed in young AF and NP cells incubated with a wide range of 17β-estradiol concentrations, revealing no cytotoxicity in either bovine nor human disc cells.
Subsequently, we investigated the potential antioxidant activity of selected 17β-estradiol concentrations in bovine AF and NP cells. Its effect on basal and H2O2-stimulated ROS levels was studied and results from the DCFH-DA assay revealed a tendency of H2O2-induced ROS levels to decrease.
We then followed by assessing the effect of 17β-estradiol on GPER receptor’s expression levels in young AF cells after incubation for 48 h with the following concentrations: 0, 1, 10, 100 nM and 1 μM. Based on our results, no significant change in GPER expression was found at the gene level, but it incubation of AF cells with 1 μM 17β-estradiol for 48 hours resulted in the up-regulation of the gene encoding type XII collagen.
Similar experiments were also performed in young NP cells to investigate the effect of 17β-estradiol on GPER expression levels. It was found that the expression pattern of the receptor did not change significantly for any of the 17β-estradiol concentrations tested.
The study of the effect of 17β-estradiol on GPER estrogen receptor’s expression levels was then extended to senescent intervertebral disc cells to investigate the role of 17β-estradiol in tissue degeneration. Incubation of bovine senescent AF cells with 1 μM 17β-estradiol resulted in a decrease of GPER transcriptional levels after 48 h of incubation, as well as in the downregulation of genes encoding metalloproteases MMP-3 and -9.
The same experiments were carried out in senescent NP cells, in which no change in GPER gene expression levels was observed. However, a significant up-regulation of the gene encoding aggrecan was observed after 48 hours of incubation with 1 μM 17β-estradiol.
Finally, the potential antioxidant activity of 17β-estradiol was investigated in senescent AF and NP cells after incubation at the concentrations of 0, 1, 10, 100 nM and 1 μM for seven days. Indeed, a dose-dependent tendency to decrease intracellular ROS levels was found in senescent NP cells. In contrast, no effect was observed on senescent AF cells.
In conclusion, the present study presents evidence that 17β-estradiol shows a protective effect against intervertebral disc degeneration by restraining the catabolic phenotype of senescent AF and NP cells and trhough its antioxidant activity on senescent NP cells. Our findings may contribute to further elucidation of the importance of 17β-estradiol in the treatment of intervertebral disc degeneration, taking into account the parameter of cellular senescence, and may therefore aid in the design of more targeted and efficient therapeutic approaches for the treatment of low back pain.
Keywords:
Low back pain, Menopause, Intervertebral disc, Annulus fibrosus (AF), Nucleus pulposus (NP), Degeneration, GPER, 17β-estradiol (E2), Estrogen receptors, SASP, ECM
