FLI1 and ERG protein degradation is regulated via Cathepsin B lysosomal pathway in human dermal microvascular endothelial cells

Abstract Objectives Friend leukemia integration 1 and erythroblast transformation‐specific, important regulators of endothelial cell homeostasis, are reduced in microvascular endothelial cells in scleroderma patients, and their deficiency has been implicated in disease pathogenesis. The goal of this study was to identify the mechanisms involved in the protein turnover of friend leukemia integration 1 and erythroblast transformation‐specific in microvascular endothelial cells. Methods The effects of lysosome and proteosome inhibitors on friend leukemia integration 1 and erythroblast transformation‐specific levels were assessed by Western blotting and capillary morphogenesis. The effect of scleroderma and control sera on the levels of friend leukemia integration 1 and erythroblast transformation‐specific was examined. Results The reduction in the protein levels of friend leukemia integration 1 and erythroblast transformation‐specific in response to interferon α or Poly:(IC) was reversed by blocking either lysosomal (leupeptin and Cathepsin B inhibitor) or proteosomal degradation (MG132). MG132, leupeptin or CTSB‐(i) also counteracted the anti‐angiogenic effects of Poly:(IC) or interferon α. Scleroderma sera reduced protein levels of friend leukemia integration 1 and erythroblast transformation‐specific in comparison to control sera. Treatment with CTSB(i) increased the levels of friend leukemia integration 1 and erythroblast transformation‐specific in a majority of serum‐treated samples. Conclusions Inhibition of cathepsin B was effective in reversing the reduction of friend leukemia integration 1 and erythroblast transformation‐specific protein levels after treatment with interferon α or scleroderma sera, suggesting that targeting cathepsin B may have a beneficial effect in SSc vascular disease.


| INTRODUC TI ON
Friend leukemia integration 1 (FLI1) and erythroblast transformation-specific (ERG) belong to the E-twenty-six (ETS) specific transcription factors family and bind to a consensus DNA sequence centered on the core GGA (A/T) motif through a helix-loop-helix domain. Friend leukemia integration 1 has been shown to play a major role in hematopoiesis, embryonic development, and vasculogenesis. 1 Friend leukemia integration 1 deficiency induced SSclike phenotypes in various cell types, including dermal fibroblasts, dermal microvascular endothelial cells (MVECs), and perivascular inflammatory cells. [2][3][4] It has been proposed, that epigenetic downregulation of FLI1 expression contributes to the profibrogenic phenotype of SSc fibroblasts. 5 The protein levels of ERG, as well as FLI1, are also reduced in SSc pulmonary vasculature. 6 Furthermore, endothelial FLI1 deficiency reproduced histopathological and functional abnormalities characteristic of SSc fibrosis and vasculopathy in animal models. 2,7,8 Unlike FLI1, which is widely expressed, ERG is more specifically expressed in endothelial and hematopoietic cells, where it functions in a manner similar to FLI1. 9 Erythroblast transformation-specific and FLI1 have been shown to cooperatively regulate vascular inflammation and EndoMT, 6,10,11 as well as endoglin gene expression. 12 Treatment of endothelial cells with proinflammatory stimuli, including LPS, TNF-α or hypoxia, downregulated ERG expression. 13 Previous studies have established that FLI1 is primarily regulated at the protein level. [14][15][16][17] While the ubiquitin-proteasome pathway has been associated with the turnover of several of the ETS family members, including ERG, [18][19][20] it is not known whether FLI1 is degraded by this mechanism, particularly in SSc.
The second major protein degradation pathway is lysosomal proteolysis. Lysosomes are ubiquitous organelles which contain approximately 50 soluble hydrolases capable of degrading various macromolecules, including proteins, lipids, and carbohydrates. 21 Lysosomes perform complex functions including endocytic, phagocytic, and autophagic degradation, antigen presentation, killing of target cells by cytotoxic T-cells and NK cells, cell adhesion and migration, tumor invasion and metastasis, plasma membrane repair, and protein degradation. 22,23 Of special interest are the members of the papain family, cysteine proteases cathepsins. 24,25 Various cathepsins are involved in lysosomal protein recycling and in several physiological processes such as antigen (Ag) processing, wound healing, bone remodeling, prohormone, and proenzyme activation, as well as in pathological conditions including cancer, bronchial asthma, atherosclerosis, periodontitis, rheumatoid arthritis (RA), and osteoarthritis. [26][27][28] Relevant to our study, it was demonstrated that upregulation of endothelial Cathepsin B enzyme (CTSB) may contribute to the development of SSc vasculopathy, especially to digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc. 29 This study aimed to examine the contribution of the proteasome and lysosome to FLI1 and ERG turnover in MVECs. We showed that both the proteasome and lysosome contribute to the degradation of FLI1 and ERG. Further, we identified CTSB as a key lysosomal enzyme responsible for degradation of FLI1 and ERG.

| Patients and controls
Serum samples were obtained from patients with limited SSc (n = 2), SSc sine scleroderma 1 or diffuse SSc (n = 7) (median age 48 years, range 25-71 years), 30 and from 10 age-matched and sex-matched healthy individuals. Patient characteristics are included in Table 1.
Prior to participation, all subjects provided written informed consent according to the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of Boston University (H-31479). Peripheral blood samples were collected without any additive, left to clot for 30 minutes before centrifugation at 1500 g for 15 minutes, and serum was collected and stored in aliquots at −80°C until used.

| Cell cultures
Microvascular endothelial cells isolated from human foreskin were cultured at 37°C in a humidified atmosphere with 5% CO 2 on bovine collagen-coated T25 flasks as previously described 31,32 in Endothelial Cell Growth Medium supplemented with 5% fetal bovine serum (FBS), 5 ng/mL H-epidermal growth factor, 1 μg/mL hydrocortisone acetate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B, without addition of further angiogenic growth factors (MV2 kit, catalog number C-22121. PromoCell Germany). The culture medium was changed every two days, and after 1 week, a monolayer of primary culture cells with small colonies of polygonal elements was detected, and using immunomagnetic beads recognizing E-Selectin, they were further identified as MVECs. Following, a second selection with CD31 immunobeads, MVECs were maintained in Endothelial Cell Growth complete Medium and were used between the second and the fourth passages in culture.
In order to find the correct concentration at which the cells result

| Statistical analysis
Data are expressed as the mean ± SEM. ANOVA and Tukey's correction multiple comparisons or Student's t test were used where appropriate for statistical evaluation of the differences between independent groups. A P value <.05 was considered statistically significant.

| Protein turnover of FLI1 and ERG in MVECs is mediated by the proteasome and lysosome
In order to determine whether the proteasome or lysosome are involved in the steady-state protein turnover of FLI1 and ERG, MVECs were treated with commonly used proteasome (MG132) or lysosome (Leupeptin) inhibitors. As displayed in Figure 1A, treatment with 100 nM of MG132 for 1, 3, and 6 hours, significantly increased FLI1 protein levels compared with each control, with a maximal increase at 3 hours ( Figure 1B), and the increased protein levels were sustained up to 24 hours (Figure 2A,B). Under the same experimental conditions, maximal increase of ERG protein levels occurred at 1 hour and persisted for 3 hours, with a smaller increase at 6 hours ( Figure 1A,C) which was maintained up to 24 hours (Figure 2A,B).
Likewise, inhibition of the lysosomal pathway by leupeptin (10 μM) for 1, 3, 6, and 24 hours showed a significant increase of FLI1 protein compared with each control (Figures 1D,E and 2A,B). Treatment with leupeptin also resulted in increased levels of ERG protein ( Figure 1D,F), which extended for up to 24 hours (Figure 2A,C).
Inhibition of lysosome or proteasome degradation had comparable effects on the levels of FLI1 and ERG proteins (Figure 2A,B).
Simultaneous blockade of lysosome and proteasome was comparable to the addition of each inhibitor alone. Since previous work has shown that eNOS degradation is regulated by the proteosomal pathway in bovine pulmonary artery endothelial cells, 33 we also assessed the protein levels of eNOS in our experimental system. We

| CTSB regulates lysosomal degradation of FLI1 and ERG proteins
Considering that leupeptin is capable of inhibiting several lysosomal enzymes, and the levels of CTSB were increased in dermal blood vessels in vivo in SSc skin, 29  of FLI1 and ERG protein levels, that was comparable to that of leupeptin at all time points tested ( Figure 3B,C). These data demonstrate that cathepsin B is a primary lysosomal enzyme responsible for degradation of FLI1 and ERG in MVECs.

| Inhibition of the proteasome and lysosome reverses IFNα-mediated FLI1 and ERG downregulation
Activation of type I interferons plays a key role in SSc pathogenesis. 34

| Inhibition of the proteasome or lysosome restores capillary morphogenesis in MVECs
To investigate the functional influence mediated by the proteasome and lysosome pathways on in vitro angiogenesis, we em- Together, these data suggest that the anti-angiogenic pathways activated by IFNα could be modulated through the regulation of the proteasome or lysosomal activity, and in particular by regulating the action of CTSB enzyme.

| CTSB inhibition reverses the inhibitory effect of SSc patient sera on FLI1 and ERG protein levels
Previous studies have shown that treatment with SSc serum reduced FLI1 protein levels in MVECs. 14, 15 We confirmed these findings in our study ( Figure 6A,B). We also showed that similar to FLI1, the levels of ERG were reduced in MVECs upon the treatment with SSc sera (Figure 6A,C). Given that circulating levels of cathepsin B are elevated in SSc sera, 29  with ERG being somewhat more responsive to the treatment than FLI1 ( Figures 6D-F and S1). Together, these data suggest that the modulation of CTSB activity could partially ameliorate the harmful effects of SSc sera on endothelial cells.  is needed before such compounds could be safely used in clinic.
If such drugs would become available, they could be very helpful for treatment of vascular diseases associated with ERG and Fli1 deficiency.

| PER S PEC TIVE S
We report that lysosomal enzyme cathepsin B plays a central role in regulating FLI1 and ERG turnover in MVECs. Further, we showed that SSc sera downregulate FLI1 and ERG proteins and that the inhibition of cathepsin B has the ability to reverse these effects.
Targeting cathepsin B could represent an attractive strategy for vascular disease in SSc. and design of the study, data analysis, and interpretation, drafted the manuscript, and gave final approval.

DATA AVA I L A B I L I T Y S TAT E M E N T
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.