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E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

A few ubiquitin ligases have been shown to target Runx2, the key osteogenic transcription factor and thereby regulate bone formation. The regulation of Runx2 expression and function are controlled both at the transcriptional and posttranslational levels. Really interesting new gene (RING) finger ubiquitin ligases of which RNF138 is a member are important players in the ubiquitin-proteasome system, contributing to the regulation of protein turnover and cellular processes. Here, we demonstrated that RNF138 negatively correlated with Runx2 protein levels in osteopenic ovariectomized rats which implied its role in bone loss. Accordingly, RNF138 overexpression potently inhibited osteoblast differentiation of mesenchyme-like C3H10T1/2 as well primary rat calvarial osteoblast (RCO) cells in vitro, whereas overexpression of catalytically inactive mutant RNF138Δ18-58 (lacks RING finger domain) had mild to no effect. Contrarily, RNF138 depletion copiously enhanced endogenous Runx2 levels and augmented osteogenic differentiation of C3H10T1/2 as well as RCOs. Mechanistically, RNF138 physically associates within multiple regions of Runx2 and ubiquitinates it leading to its reduced protein stability in a proteasome-dependent manner. Moreover, catalytically active RNF138 destabilized Runx2 which resulted in inhibition of its transactivation potential and physiological function of promoting osteoblast differentiation leading to bone loss. These findings underscore the functional involvement of RNF138 in bone formation which is primarily achieved through its modulation of Runx2 by stimulating ubiquitin-mediated proteasomal degradation. Thus, our findings indicate that RNF138 could be a promising novel target for therapeutic intervention in postmenopausal osteoporosis., (© 2024 Wiley Periodicals LLC.)

Ormeloxifene (ORM) (3,4‐trans‐2,2‐dimethyl‐3‐phenyl‐4‐p‐(β‐pyrrolidinoethoxy) phenyl‐7‐methoxychroman), world's first nonsteroidal selective estrogen receptor modulator approved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORM can induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan‐caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c‐Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h, respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells. [ABSTRACT FROM AUTHOR]