Regulation of p27 and Cdk2 Expression in Different Adipose Tissue Depots in Aging and Obesity

Aging usually comes associated with increased visceral fat accumulation, reaching even an obesity state, and favoring its associated comorbidities. One of the processes involved in aging is cellular senescence, which is highly dependent on the activity of the regulators of the cell cycle. The aim of this study was to analyze the changes in the expression of p27 and cdk2 in different adipose tissue depots during aging, as well as their regulation by obesity in mice. Changes in the expression of p27 and CDK2 in visceral and subcutaneous white adipose tissue (WAT) biopsies were also analyzed in a human cohort of obesity and type 2 diabetes. p27, but not cdk2, exhibits a lower expression in subcutaneous than in visceral WAT in mice and humans. p27 is drastically downregulated by aging in subcutaneous WAT (scWAT), but not in gonadal WAT, of female mice. Obesity upregulates p27 and cdk2 expression in scWAT, but not in other fat depots of aged mice. In humans, a significant upregulation of p27 was observed in visceral WAT of subjects with obesity. Taken together, these results show a differential adipose depot-dependent regulation of p27 and cdk2 in aging and obesity, suggesting that p27 and cdk2 could contribute to the adipose-tissue depot’s metabolic differences. Further studies are necessary to fully corroborate this hypothesis.

SIRT7-Induced PHF5A Decrotonylation Regulates Aging Progress Through Alternative Splicing-Mediated Downregulation of CDK2

Dysregulation of protein posttranslational modification (PTM) can lead to a variety of pathological processes, such as abnormal sperm development, malignant tumorigenesis, depression, and aging process. SIRT7 is a NAD+-dependent protein deacetylase. Besides known deacetylation, SIRT7 may also have the capacity to remove other acylation. However, the roles of SIRT7-induced other deacylation in aging are still largely unknown. Here, we found that the expression of SIRT7 was significantly increased in senescent fibroblasts and aged tissues. Knockdown or overexpression of SIRT7 can inhibit or promote fibroblast senescence. Knockdown of SIRT7 led to increased pan-lysine crotonylation (Kcr) levels in senescent fibroblasts. Using modern mass spectrometry (MS) technology, we identified 5,149 Kcr sites across 1,541 proteins in senescent fibroblasts, and providing the largest crotonylome dataset to date in senescent cells. Specifically, among the identified proteins, we found SIRT7 decrotonylated PHF5A, an alternative splicing (AS) factor, at K25. Decrotonylation of PHF5A K25 contributed to decreased CDK2 expression by retained intron (RI)-induced abnormal AS, thereby accelerating fibroblast senescence, and supporting a key role of PHF5A K25 decrotonylation in aging. Collectively, our data revealed the molecular mechanism of SIRT7-induced k25 decrotonylation of PHF5A regulating aging and provide new ideas and molecular targets for drug intervention in cellular aging and the treatment of aging-related diseases, and indicating that protein crotonylation has important implications in the regulation of aging progress.

Osthole Alleviates Neointimal Hyperplasia in Balloon-Induced Arterial Wall Injury by Suppressing Vascular Smooth Muscle Cell Proliferation and Downregulating Cyclin D1/CDK4 and Cyclin E1/CDK2 Expression

Percutaneous coronary intervention (PCI) is the most widely used therapy for treating ischemic heart disease. However, intimal hyperplasia and restenosis usually occur within months after angioplasty. Modern pharmacological researchers have proven that osthole, the major active coumarin of Cnidium monnieri (L.) Cusson, exerts potent antiproliferative effects in lung cancer cells, the human laryngeal cancer cell line RK33 and TE671 medulloblastoma cells, and its mechanism of action is related to cell cycle arrest. The goal of the present study was to observe the effect of osthole on vascular smooth muscle cell (VSMC) proliferation using platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMCs isolated from rats and vascular balloon injury as models to further elucidate the molecular mechanisms underlying this activity. We detected the relative number of VSMCs by the MTT assay and EdU staining and examined cell cycle progression by flow cytometry. To more deeply probe the mechanisms, the protein expression levels of PCNA, the cyclin D1/CDK4 complex and the cyclin E1/CDK2 complex in balloon-treated rat carotid arteries and the mRNA and protein expression levels of the cyclin D1/CDK4 and cyclin E1/CDK2 complexes in VSMCs were detected by real-time RT-PCR and western blotting. The data showed that osthole significantly inhibited the proliferation of VSMCs induced by PDGF-BB. Furthermore, osthole caused apparent VSMC cycle arrest early in G0/G1 phase and decreased the expression of cyclin D1/CDK4 and cyclin E1/CDK2. Our results demonstrate that osthole can significantly inhibit PDGF-BB-induced VSMC proliferation and that its regulatory effects on cell cycle progression and proliferation may be related to the downregulation of cyclin D1/CDK4 and cyclin E1/CDK2 expression as well as the prevention of cell cycle progression from G0/G1 phase to S phase. The abovementioned mechanism may be responsible for the alleviation of neointimal hyperplasia in balloon-induced arterial wall injury by osthole.

Immunohistochemical Study of NR2C2, BTG2, TBX19, and CDK2 Expression in 31 Paired Primary/Recurrent Nonfunctioning Pituitary Adenomas

This study investigated potential markers for predicting nonfunctioning pituitary adenoma (NFPA) invasion and recurrence by high-throughput tissue microarray analyses. We retrospectively studied two groups of patients: 60 nonrecurrent NFPA cases that included noninvasion and invasion subtypes and 43 recurrent cases that included primary NFPA. A total of 31 paired patient samples were evaluated (12 patients with one surgery and 31 who had undergone two operations, with both tumors analyzed). Expressions of nuclear receptor subfamily 2 group C member 2 (NR2C2), B cell translocation gene 2, T-box-19 (TBX19), and cyclin-dependent kinase 2 (CDK2) in surgically resected specimens were assessed by immunohistochemistry. The relationships between marker expression and clinical characteristics including age, sex, tumor volume, and follow-up time were analyzed. Tumor volume and invasion as well as follow-up time were significantly associated with invasion and recurrence (P < 0.01). Of the 60 nonrecurrent samples, 15/41 and 13/19 showed high NR2C2 expression in the noninvasion and invasion groups, respectively (χ2 =5.287, P = 0.021). NR2C2 was also overexpressed in 43 primary recurrent cases (χ2 =5.433, P = 0.02), whereas CDK2 (χ2 = 11.242, P = 0.001) and TBX19 (χ2 = 4.875, P = 0.027) were downregulated. In the 31 paired samples, NR2C2 was more highly expressed in the recurrent as compared to the primary tumor. High NR2C2 expression was associated with NFPA invasion, recurrence, and progression, while TBX19 and CDK2 were associated with NFPA recurrence.

Heme Oxygenase-1, Ferroportin and SAMHD1 Induction By Heme Inhibits HIV-1

Background Heme oxygenase-1 (HO-1) induction by hemin inhibitsHIV-1 infection in cultured macrophages and T-cells and also in HIV-1 infected humanized mice via a protein kinase C-dependent pathway (reviewed in [1]). LPS-treated human macrophages express HO-1 which may protect them against HIV-1 infection through the production of MIP1α, MIP1β and LD78β chemokines that decrease CCR5 expression. Iron depletion by iron chelators or through the expression of ferroportin, an iron export protein, inhibited HIV-1 [2, 3]. Objective We analyzed the effect of heme on HIV-1 infection in macrophages and the contribution of heme and iron regulatory proteins including HO-1, ferroportin and hepcidin and cell cycle regulatory cell cycle dependent kinase 2 (CDK2) and p21, which are deregulated by iron depletion. We also analyzed SAM domain and HD domain-containing protein 1 (SAMHD1) as it was shown to be regulated by CDK2 and p21. Methods One round HIV-1 infection was analyzed in THP-1 cells infected with VSVg-pseudotyped HIV-1 expressing luciferase. Gene expression and protein expression analysis was carried out using RNA and protein isolated from hemin treated and untreated cells using RT PCR western blot respectively. Specific gene knock-downswere generated in THP-1 cells using lentivirus expressing gene-targeted shRNAs. Results In heme-treated THP-1 cells, mRNA expression of ferroportin, p21, SAMHD1, hypoxia-induced factor (HIF)-1α, and IKBα and NF-κB inhibitor was increased and CDK2 expression decreased. Stable ferroportin knock-down prevented HIV-1 inhibition in THP-1 cells. Treatment with hepcidin also restored HIV-1 inhibition in hemin treated THP-1 cells. Ferritin levels increased in Hemin treated THP1 cells and decreased when cells were treated with tin protoporphyrin (SnPP) IX inaddtion to hemin, suggesting that HO-1 induction leads to intracellular iron accumulation. In addition, hemin treatment reduced the expression of p65 subunit of NF-kB and induced expression of NF-κB inhibitor, IKBα. We also observed increased expression of p21 and decreased expression of CDK2 in the cells treated with hemin and accumulation of the cells in the G1 phase of the cell cycle. Both CDK2 and p21 control phosphorylation of SAMHD1 which in turn controls the dNTP pool and prevents HIV-1 reverse transcription. We observed reduced expression of CDK2 and increased expression of SAMHD1 and p21 in hemin treated cells, which may also contribute to the inhibition of HIV-1. Stable HIF-1a knockdown in THP-1 cells increased HIV-1 replication indicating that HIF-1a might also restrict HIV-1 replication. Conclusion Our study shows that induction of HIF-1a and iron export and utilization proteins protects hemin-treated THP-1 cells from HIV-1 infection. Block of HIV-1 inhibition by heme in THP-1 cells with stable ferroportin knock-down and restoration of HIV-1 replication with hepcidin in heme-treated THP-1 cells suggest that ferroportin plays a key role in the HIV-1 inhibition. Additional molecular mechanisms of heme-mediated HIV-1 inhibition might also include NF-kB inhibition by IKBα, reduction of CDK2 expression and induction of SAMHD1 and p21. Thus this complex deregulation of iron metabolism leads to the inhibition of HIV-1 transcription. Acknowledgments This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, U19AI109664 and 5G12MD007597) and District of Columbia Developmental Center for AIDS Research grant (1P30AI117970). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References 1. Nekhai S, Kumari N, Dhawan S: Role of cellular iron and oxygen in the regulation of HIV-1 infection. Future Virol 2013, 8(3):301-311. 2. Kumari N, Iordanskiy S, Kovalskyy D, Breuer D, Niu X, Lin X, Xu M, Gavrilenko K, Kashanchi F, Dhawan S et al: Phenyl-1-Pyridin-2yl-ethanone-based iron chelators increase IkappaB-alpha expression, modulate CDK2 and CDK9 activities, and inhibit HIV-1 transcription. Antimicrob Agents Chemother 2014, 58(11):6558-6571. 3. Xu M, Kashanchi F, Foster A, Rotimi J, Turner W, Gordeuk VR, Nekhai S: Hepcidin induces HIV-1 transcription inhibited by ferroportin. Retrovirology 2010, 7:104. Disclosures No relevant conflicts of interest to declare.

Epoetin Alpha and Epoetin Zeta: A Comparative Study on Stimulation of Angiogenesis and Wound Repair in an Experimental Model of Burn Injury

Deep second-degree burns are characterized by delayed formation of granulation tissue and impaired angiogenesis. Erythropoietin (EPO) is able to stimulate angiogenesis and mitosis, activating vascularization and cell cycle. The aim of our study was to investigate whether two biosimilar recombinant human erythropoietins, EPO-αand EPO-Z, may promote these processes in an experimental model of burn injury. A total of 84 mice were used and a scald burn was produced on the back after shaving, in 80°C water for 10 seconds. Mice were then randomized to receive EPO-α(400 units/kg/day/sc) or EPO-Z (400 units/kg/day/sc) or their vehicle (100 μL/day/sc 0.9% NaCl solution). After 12 days, both EPO-αand EPO-Z increased VEGF protein expression. EPO-αcaused an increased cyclin D1/CDK6 and cyclin E/CDK2 expression compared with vehicle and EPO-Z (p<0.001). Our study showed that EPO-αand EPO-Z accelerated wound closure and angiogenesis; however EPO-αresulted more effectively in achieving complete skin regeneration. Our data suggest that EPO-αand EPO-Z are not biosimilars for the wound healing effects. The higher efficacy of EPO-αmight be likely due to its different conformational structure leading to a more efficient cell proliferation and skin remodelling.