Heat shock protein is a unique marker of growth arrest during macrophage differentiation of HL-60 cells

Prior to morphologic and functional maturation, terminally differentiating hematopoietic cells first exit the cell cycle and undergo growth arrest. Relatively little is known about which molecules regulate differentiation-induced growth arrest. In the present report, we sought to determine whether the mammalian low molecular weight heat shock protein (hsp28) was a candidate growth-regulatory molecule during human hematopoiesis. To this end, hsp28 protein expression was examined during phorbol ester (PMA)-induced macrophage differentiation of the human HL-60 promyelocytic leukemic cell line. Whereas hsp28 was constitutively expressed at relatively low levels in an unphosphorylated state, hsp28 was rapidly phosphorylated within 4 hr following PMA-induced differentiation, preceding increased hsp28 protein levels at 24–48 h. In contrast to other differentiative agents, hsp28 steady state mRNA and protein were regulated concordantly in response to macrophage differentiation. More importantly, these changes were transient, and occurred concomitant with the down-regulation of cellular proliferation and the onset of G₁ phase cell cycle arrest. In total, these observations implicate hsp28 as an intermediary in the myelomonocytic differentiative pathway of promyelocytic leukemic cells, and will shed light on the events regulating this process. © 1993 Wiley-Liss, Inc.     

A protein complex expressed during terminal differentiation of monomyelocytic cells is an inhibitor of cell growth

A protein complex (PC) composed of the MRP8 and MRP14 proteins has previously been shown to be a specific inhibitor of casein kinase I and II. This PC is expressed during the late stages of terminal differentiation induced in human promyelocytic HL-60 leukemia cells by 1 alpha,25-dihydroxyvitamin D3 and in human monocytic THP-1 leukemia cells by phorbol 12-myristate 13-acetate. This expression is associated with terminal cell differentiation because incubation of HL-60 cells with an agent or condition that causes suppression of growth but not induction of differentiation does not result in expression of the PC. At concentrations of 5-15 nM, the purified PC inhibited the growth of HL-60 cells and THP-1 cells, as well as other cell types belonging to different cell lineages. This growth inhibition was preceded by a reduction in [32P]phosphate incorporation and, at the higher PC concentrations, was associated with a reduction in [3H]thymidine, [3H]uridine, and [32S]methionine incorporation. The specific expression pattern and growth-inhibitory character of the PC suggests that the complex may have a role in suppressing cell growth during monomyelocytic terminal differentiation induced by specific chemical stimuli and during physiological and pathological events associated with monomyelocytic cell functions.     

A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila

In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3–dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3_Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3_Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis–like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation.     

Rise and fall of crystallin gene messenger levels during fibroblast growth factor induced terminal differentiation of lens cells.

Explanted rat lens epithelial cells differentiate synchronously in vitro to lens fiber cells in the presence of basic fibroblast growth factor (bFGF). We have monitored the expression of the three rat crystallin gene families, the alpha-, beta-, and gamma-crystallin genes, during this process. The expression of these gene families is sequentially activated, first the alpha-crystallin genes at Day 1, then the beta-crystallin genes at Day 3, and finally the gamma-crystallin genes at Day 8. The steady state levels of alpha- and beta-crystallin mRNA are not affected by incubation with actinomycin D, suggesting that these mRNAs are stable. Nevertheless, all crystallin mRNAs disappear from the differentiated explants between Days 10 and 11, a process signaled by bFGF. At this time a novel abundant mRNA appears. Cloning and sequencing showed that this mRNA encoded aldose reductase. Our results suggest a novel model for the regulation of crystallin synthesis during lens cell differentiation: a gene pulse delivers a certain amount of stable mRNA, this mRNA is removed at a later stage of differentiation by a stage-specific breakdown mechanism. Each of these regulatory steps requires a signal from bFGF.     

Autocrine beta-related interferon controls c-myc suppression and growth arrest during hematopoietic cell differentiation

Different hematopoietic cells produce minute amounts of beta-related interferon (IFN) following induction of differentiation by chemical or natural inducers. The endogenous IFN binds to type I cell surface receptors and modulates gene expression in the producer cells. We show that self-induction of two members of the IFN-induced gene family differs in the dose response sensitivity and the prolonged kinetics of mRNA accumulation from the response to exogenous IFN-beta 1. Production and response to endogenous IFN are also detected when bone marrow precursor cells differentiate to macrophages after exposure to colony stimulating factor 1. In M1 myeloid cells induced to differentiate by lung-conditioned medium, addition of antibodies against IFN-beta partially abrogates the reduction of c-myc mRNA and the loss in cell proliferative activity, which both occur during differentiation. The endogenous IFN therefore functions as an autocrine growth inhibitor that participates in controlling c-myc suppression and the specific G0/G1 arrest during terminal differentiation of hematopoietic cells.     

Regulation of the M-cadherin-beta-catenin complex by calpain 3 during terminal stages of myogenic differentiation

The cysteine protease calpain 3 (CAPN3) is essential for normal muscle function, since mutations in CAPN3 cause limb girdle muscular dystrophy type 2A. Previously, we showed that myoblasts isolated from CAPN3 knockout (C3KO) mice were able to fuse to myotubes; however, sarcomere formation was disrupted. In this study we further characterized morphological and biochemical features of C3KO myotubes in order to elucidate a role for CAPN3 during myogenesis. We showed that cell cycle withdrawal occurred normally in C3KO cultures, but C3KO myotubes have an increased number of myonuclei per myotube. We found that CAPN3 acts during myogenesis to specifically control levels of membrane-associated but not cytoplasmic beta-catenin and M-cadherin. CAPN3 was able to cleave both proteins, and in the absence of CAPN3, M-cadherin and beta-catenin abnormally accumulated at the membranes of myotubes. Given the role of M-cadherin in myoblast fusion, this finding suggests that the excessive myonuclear index of C3KO myotubes was due to enhanced fusion. Postfusion events, such as beta1D integrin expression and myofibrillogenesis, were suppressed in C3KO myotubes. These data suggest that the persistence of fusion observed in C3KO cells inhibits subsequent steps of differentiation, such as integrin complex rearrangements and sarcomere assembly.     

CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation.     

Transforming growth factor beta regulates the inhibitory actions of epidermal growth factor during granulosa cell differentiation

The effects of transforming growth factor beta (TGF-beta) on epidermal growth factor (EGF) receptor content and EGF action were studied in cultured granulosa cells from immature diethylstilbestrol-implanted rats. During follicle-stimulating hormone (FSH)-induced differentiation in vitro, EGF receptors increased by 20-fold as measured by the binding of 125I-EGF to the intact cells. Addition of TGF-beta during the 48-h culture period amplified the stimulatory effects of FSH on EGF receptors up to 2-fold, with ED50 and maximal concentrations of 2.5 and 8 pM, respectively. Also TGF-beta alone in amounts from 1.6 to 16 pM increased EGF receptor content 4-fold. The stimulatory effects of TGF-beta were due to increased numbers of EGF receptors/cell, since the growth factor had no effect on the Kd (3-5 X 10(-11) M) of the high-affinity EGF binding site. TGF-beta action was observed within 20 h of granulosa cell culture and was maximal by 48 h of a 96-h culture. The stimulatory actions of TGF-beta in gonadotropin-induced cells were exerted through the cAMP effector system of the granulosa cell, since the growth factor also amplified the induction of EGF receptors by cholera toxin, forskolin, and 8-bromo-cAMP. The augmentation of EGF receptors by TGF-beta resulted in a parallel 2-fold increase in the inhibitory effects of EGF on FSH-induced cAMP production and luteinizing hormone receptor expression during granulosa cell development. TGF-beta did not increase granulosa cell numbers during culture although it elevated [3H]thymidine incorporation into DNA by 2-fold over that of FSH-treated cells. These results indicate that TGF-beta regulates the effects of both FSH and EGF during granulosa cell differentiation and provides evidence that ovarian function may be controlled by the combined actions of gonadotropins and multiple growth factors.