Abolished adherence alters signaling pathways in phorbol ester-induced human U937 cells

Abstract

Phorbol ester (TPA) treatment of human U937 myeloid leukemia cells is associated with increasing adherence and monocyte-like maturation whereby the role of β2 integrin-mediated attachment for subsequent growth properties and the differentiation program remains unclear. Here, stably-transfected U937 cells with a pMTH1 vector containing the β2 integrin gene of CD11b in antisense orientation (asCD11b-U937) demonstrated a significantly reduced proliferative capacity in contrast to control vector transfectants (pMTH1-U937) or wild-type U937 cells. Phorbol ester exposure induced adherence and growth arrest in more than 90% of pMTH1-U937 and wild-type U937 cells after 72 h. In contrast, TPA-treated asCD11b-U937 failed to attach and the proliferation continued in more than 30% of the cells. Moreover, increased apoptosis appeared in asCD11b-U937 after TPA induction in contrast to pMTH1-U937 cells. In addition, non-specific inhibition of adherence on an agarose surface demonstrated internucleosomal DNA fragmentation in both, pMTH1-U937 and asCD11b-U937 after TPA treatment indicating a functional relationship between abolished adherence, regulation of proliferation and induction of apoptosis. Western blot analysis revealed differences in the expression levels and altered phosphorylation patterns of Pyk-2, pp60src and p42/p44 MAP kinases between pMTH1-U937 and asCD11b-U937 following TPA exposure which was also substantiated by Pyk-2 immunoprecipitation. These findings suggested that induced adherence predominantly mediated by a functional CD11b/CD18 integrin in U937 cells is involved in the activation of downstream signaling kinases and contributes to cell cycle regulation and apoptosis during monocytic maturation.

Involvement of CD11b integrin in the alteration of metabolic factors after phorbol ester stimulation of human myeloid leukemia cells

“…The proliferation of wild-type U937 and pMTH1-U937 cells as measured by 3  H]thymidine incorporation revealed a significant reduction by about 75% to 85% after 72 h of TPA treatment (Figure 1b) which is in agreement with previous studies demonstrating TPA-mediated growth inhibition [1,10,11]. In contrast, phorbol ester exposure to asCD11b-U937 cells was associated with a sustained 3 H]thymidine incorporation in more than 80% of the cells (Figure 1b).…”

Kidney Injury Accelerates Cystogenesis via Pathways Modulated by Heme Oxygenase and Complement

“…14 Local, rather that systemic, regulation of the C3-mediated responses in cystic kidneys is suggested by the strong localization of C3 into cystic and noncystic renal tubular cells in ARPKD and Cys1 cpk/cpk kidneys, 14 by the capacity of the renal tubular epithelium to produce as well as activate C3 14 and by the suggested role of fluid flow force in this regulation (Figures 4 and 5). Also, C3 plays a central role in differentiation, attachment and survival of monocytes/macrophages, 42,43 and their M2-polarization 44 (a hallmark feature of accelerated cystogenesis in the Cys1 cpk model of ARPKD 14 ), and depletion of macrophages by liposomal clodronate attenuates cystogenesis in orthologous ADPKD models by reducing proliferation of cystic tubules. 45 In addition to the macrophage effects, C3 may also induce procystogenic 30,46 TNF release 47 and possibly directly activate c-Src 48 in renal tubule cells expressing CR3 receptors (e.g., proximal tubule and collecting duct cells; data not shown).…”

Integrins in the Spotlight of Cancer

“…The most prominent example might be the cell treatment with the plant- derived compound phorbol ester. Upon treatment with this substance, promonocytic cell lines induced cellular adherence and growth inhibition via upregulation of the β2 integrin subunit [96]. Fish oil fatty acids (e.g., ω3 and ω6 polyunsaturated fatty acids) were shown to inhibit in vitro integrin-mediated proliferation by repressing ILK in human lung cancer [97].…”