PHLPP1 deficiency protects against age‐related intervertebral disc degeneration

Abstract

Background Intervertebral disc (IVD) degeneration is strongly associated with low back pain and is highly prevalent in the elderly population. Hallmarks of IVD degeneration include cell loss and extracellular matrix degradation. The PH domain leucine‐rich‐repeats protein phosphatase (PHLPP1) is highly expressed in diseased cartilaginous tissues where it is linked to extracellular matrix degradation. This study explored the ability of PHLPP1 deficiency to protect against age‐related spontaneous IVD degeneration. Methods Lumbar IVDs of global Phlpp1 knockout (KO) and wildtype (WT) mice were collected at 5 months (young) and 20 months (aged). Picrosirius red–alcian blue staining (PR‐AB) was performed to examine IVD structure and histological score. The expression of aggrecan, ADAMTS5, KRT19, FOXO1 and FOXO3 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human nucleus pulposus (NP) samples were obtained from patients diagnosed with IVD degeneration. PHLPP1 knockdown in human degenerated NP cells was conducted using small interfering RNA (siRNA) transfection. The expression of PHLPP1 regulated downstream targets was analyzed via immunoblot and real time quantitative PCR. Results Histological analysis showed that Phlpp1 KO decreased the prevalence and severity of age‐related IVD degeneration. The deficiency of PHLPP1 promoted the increased expression of NP phenotypic marker KRT19, aggrecan and FOXO1, and decreased levels of ADMATS5 and cell apoptosis in the NP of aged mice. In degenerated human NP cells, PHLPP1 knockdown induced FOXO1 protein levels while FOXO1 inhibition offset the beneficial effects of PHLPP1 knockdown on KRT19 gene and protein expression. Conclusions Our findings indicate that Phlpp1 deficiency protected against NP phenotypic changes, extracellular matrix degradation, and cell apoptosis in the process of IVD degeneration, probably through FOXO1 activation, making PHLPP1 a promising therapeutic target for treating IVD degeneration.

Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health

“…The FOXO transcription factors are indispensable for maintaining IVD homeostasis, and our previous findings showed that PHLPP1 depletion promoted FOXO1 expression in the NP of both mouse and human IVDs. 25,26 Similar to PHLPP1 knockdown, culture with NSC45586 increased the protein abundance of FOXO1 in degenerated human NP cells from male donors (Figure 7B). The FOXO1 expression was not affected in NP cells treated with NSC117079 (Figure 7A).…”

“…25,26 We showed that in degenerated human NP cells, PHLPP1 knockdown increased FOXO1 expression and promoted NP cell regeneration. 26 In the present study, we tested the efficacy and underlying mechanism of the two small molecule PHLPP inhibitors NSC45586 and NSC117079 in promoting NP health in mice IVDs and human NP cells by utilizing mouse ex vivo and human in vitro cultures. NP morphology, phenotype, and apoptosis were assessed to determine the protective effects of the inhibitors.…”

“…All proteins were normalized to GAPDH (Cell Signaling Technology, 884S), and band densities were calculated using imageJ as previously described. 26 NP cell culture studies. The statistical analyses were performed using GraphPad Prism9 (GraphPad Software, Inc., La Jolla, CA).…”

“…NP cells were harvested as previously described. 26 In brief, NP tissues were collected immediately after surgery and minced into small fragments ($1 mm 3 ).…”

An integrated RNA-proteomic landscape of drug induced senescence in a cancer cell line

“…Examples include PHLPP1, which protects against age-related intervertebral disc degeneration [49], and ANLN, the depletion of which induces cellular senescence [50]. Expression of ITGB4 is reportedly down-regulated under oxidative stress or upon inflammatory stimulation leading to the induction of senescence in epithelial cells, mediated through p53 activity [51].…”