Hematopoiesis is a highly and precisely regulated multistage process by which all of the different cell lineages (erythroid cells, lymphocytes and myeloid cells) that form the immune and blood systems originate from pluripotent stem cells [33, 34]. Erythropoiesis happens in human red bone marrow after kidneys responses to low levels of oxygen by releasing erythropoietin [35]. Erythropoiesis is a multi-step cellular course by which a primitive multipotent HSC experiences a series of differentiations resulting in production of erythroid lineage, undergoing erythroid progenitors (colony-forming unit erythroid [CFU-E] and burst-forming unit erythroid [BFU-E]), normoblasts, proerythroblasts, early basophilic erythroblasts, late basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, reticulocytes, ultimately differentiating to mature erythrocytes [5, 6]. Megakaryopoiesis occurs through a hierarchical series of progenitor cells, multipotent progenitor (MPP), common myeloid progenitor (CMP) and megakaryocyte-erythroid progenitor (MEP), megakaryocyte progenitor (MKP), ultimately differentiating to mature megakaryocytes [36]. The two dynamic processes are mediated by a balance of intrinsic and extrinsic factors, containing transcription factors, growth factors and miRNAs, and destruction of the two dynamic processes leads to CML. Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL for CML therapy have effectively improved the survival of CML patients, however, about 20% of CML patients have not been benefited from TKIs treatment, commonly due to TKIs resistance which lead to disease relapse and progression [37–39]. Therefore, it is urgent to seek more efficient therapeutic strategies to overcome the problem. Deeper study of the molecular mechanisms governing the development, progression and differentiation of CML can lead to finding novel therapeutic targets and improving the therapy effects for CML patients.
CRK proteins are the predominant phosphorylation substrates for BCR-ABL, which is found in over 95% of CML and 25% of acute lymphoblastic leukemias (ALL) [40]. Although CRKII and CRKL share a high degree of homology within their functional domains, CRKL is the major tyrosine-phosphorylated protein in BCR-ABL-driven CML patient neutrophils [40]. The preferential binding of BCR-ABL to CRKL, even in the presence of CRKII [41], suggests disparity in interaction properties and differential regulation of CRK proteins by BCR-ABL or ABL tyrosine kinases. These finds imply that CRKII and CRKL may play different role in CML, so in this work we investigated the exact effect of CRKII and CRKL on erythropoiesis and megakaryopoiesis of CML. The current study illustrated for the first time that CRKL but not CRKII inhibits erythroid and megakaryocyte differentiation via the inactivating Raf/MEK/ERK/Elk pathway.
CRKL deregulation is linked to the development and progression of a variety of cancers [26–28]. As we summarized in our review [25], abnormal CRKL expression is associated with gastric cancer, glioblastoma multiforme, hepatocellular carcinoma, bladder cancer, lung cancer, colon cancer, ovarian cancer, leukemia, breast cancer, head and neck cancer, rhabdomyosarcoma and neuroblastoma. It is of promise as an indicator for cancer development, invasion and metastasis as well as an attractive target for the diagnosis and prognosis of cancer. CRKL is a major tyrosine-phosphorylated protein in CML cells, pCRKL plays a special role in CML pathogenesis, and the constitutive phosphorylation of CRKL is unique to CML, which makes it a useful target for therapeutic intervention [42–44]. We previous reported that CRKL is associated with proliferation, migration and invasion of hepatocarcinoma and clear cell renal cell carcinoma cells [45–50]. However, the exact role of CRKL in CML is unknown. Our current work showed that the upregulation of CRKL potentially promotes the clinical development and progression of CML patients and enhances CML cell aggressiveness. CRKL was universally overexpressed in CML patient samples compared with normal samples (Fig. 1A). Interestingly, CRKL expression level was lower in CR patient samples than in corresponding CML patient samples (Fig. 1B). Our results indicate that CRKL is a tumor promoter playing a vital role in the development and progression of CML. To the best of our knowledge, this work is the first reporting the expression pattern of CRKL in CML patients, CR patients and normal samples. CRKⅡ deregulation is also linked to the development and progression of a variety of cancers [26–28], But our results show that CRKⅡ is only slightly overexpressed in CML (Fig. 1C, D), and that it may not play an important role in the development and progression of CML.
It is known that the CRK family plays important roles in the regulation of cell differentiation. v-CRK overexpression can increase rat pheochromocytoma PC12 cell differentiation [29], and both SH2 and SH3 domains of the CRK protein are required for neuronal differentiation of PC12 cells [30]. Moreover, CRKⅡ enhances osteoclast differentiation by activating Rac1, the overexpression of CRKⅡ and CRKL significantly enhances RANKL-induced osteoclast differentiation, and the downregulation of CRKⅡ and CRKL synergistically decreases RANKL-induced osteoclast differentiation [31]. The effect of CRKL and CRKⅡ on leukemia cell differentiation has not been reported, in our study we investigated the potential role of CRKL and CRKⅡ in erythroid and megakaryocyte differentiation of K562 cells. Hemin is an iron-containing porphyrin which is involved in oxygen delivery and used to treat acute porphyria and thalasssemia intermedia, and is also a relatively strong inducer for heme biosynthesis of K562 cell erythroid differentiation [51]. Using K562 cells as a model, we found that CRKL expression level was downregulated in hemin-induced erythroid differentiation of K562 cells (Fig. 2C), indicating CRKL might play an important role in erythroid differentiation of K562 cells. In order to verify the hypothesis, we selected previously successfully constructed CRKL stably downregulated monoclonal cell lines to investigate the effect of endogenous CRKL on erythroid differentiation. We further found that CRKL downregulation promoted erythroid differentiation of K562 cells with more benzidine-positive cells and higher mRNA expression levels of γ-globin and GPA (Fig. 3). Moreover, CRKL downregulation enhanced megakaryocyte differentiation of K562 cells with increased number of megakaryocyte cells and higher mRNA expression levels of CD41 and CD61 (Fig. 5). Our results first demonstrate CRKL is a new regulator of erythroid and megakaryocyte differentiation of K562 cells. Furthermore, we screened the differentially expressed molecules between shRNA-CRKL-K562 and shRNA-NC-K562 cells using gene microarray and iTRAQ quantitative proteomic analysis. Results showed hemoglobin molecules HBD, HBA1, HBA2, HBZ, HBE1 and HBG1 were more upregulated in shRNA-CRKL-K562 than in shRNA-NC-K562 cells (Tables 2 and 3). Moreover, GATA-1 and HMGB2 expression were increased in shRNA-CRKL-K562 than in shRNA-NC-K562 cells (Fig. 4), which are crucial for erythrocyte and megakaryocyte lineages. The zinc-finger transcription factor GATA-1 binds to GATA/AATC consensus elements in the globin gene clusters and other erythroid or megakaryocytic cell-specific genes [52]. The zinc-finger proto-oncogene Gfi-lb is an erythroid-specific transcription factor that plays an important role in erythropoiesis [53], Gfi-1B gene disruption results in embryonic death of mice due to failure to produce red blood [54]. The Gfi-1B promoter contains 2 tandem sites which includes both a GATA-1- and an Oct-1-binding sequence [55]. HMGB2 bends DNA at the Gfi-1B promoter by binding to the Gfi-1B promoter to facilitate the binding of Oct-1 to the Gfi-1B promoter [56], subsequently enhancing the binding of GATA-1 to the AATC sites of Gfi-1B promoter and activating the transcription of Gfi-1B [57]. Our results show that CRKL regulates erythroid and megakaryocyte differentiation of K562 cells by upregulating GATA-1 and HMGB2 expression.
However, the expression level of CRKⅡ was not changed in hemin-induced erythroid differentiation of K562 cells (Fig. 2D) and CRKⅡ downregulation did not affect erythroid differentiation of K562 cells (Fig. 7B). Moreover, we further investigated the effect of CRKⅡ knockdown on erythroid differentiation of K562 cells by transiently transfecting siCRKⅡ in shRNA-CRKL-K562 cells, interestingly, consistent with the above results, CRKⅡ knockdown in K562 cells with CRKL downregulation (Fig. 7F). Collectively, CRKⅡ is not associated with erythroid differentiation of K562 cells. Although CRKII and CRKL have a high degree of similarity in sequence, the two isoforms vary in ligand affinities and specificity, and the 3-dimensional structures of CRKII and CRKL differ to engage key signaling partners [21, 28]. The respective knockout mice have distinct phenotypes but both proteins are required for embryonic development [58, 59]. So CRKII and CRKL might function differently in leukemogenesis, erythropoiesis and megakaryopoiesis of CML, which deserves more attention to understand the differences between the two CRK adapter proteins. Our results are also consistent with the previous report that CRKL expression level is highest in adult hematopoietic tissues and low in epithelial tissues, whereas CRKII exhibits the highest expression in the brain, lung, kidney and low expression in bone marrow [60].
The Ras/Raf/MEK/ERK signaling pathway is involved in erythropoiesis which mainly promotes growth, differentiation and prevents apoptosis of hematopoietic cells [61–63]. MASL1 could induce erythroid differentiation in CD34 cells through the Raf/MEK/ERK signaling pathway [64]. G protein expression levels increased and ERK1/2 activated during hemin-induced differentiation of K562 cells [65]. Inhibition of the MEK/ERK signaling pathway promoted erythroid differentiation and reduced HSCs engraftment in ex-vivo expanded haematopoietic stem cells [66]. The ERK/MAPK pathway is involved in megakaryocytic differentiation of K562 cells induced by 3-hydrogenkwadaphnin [67]. Our results showed that CRKL affected the expression levels of Raf/MEK/ERK-EIk-1 pathway-related molecules (Fig. 6), we speculated CRKL might mediate erythroid and megakaryocyte differentiation of K562 cells by regulating the Raf/MEK/ERK-EIk-1 pathway. We validated the potential involvement of Raf/MEK/ERK-EIk-1 using a specific ERK inhibitor PD98059. The expression levels of GPA, γ-globin, CD41 and CD61 decreased after blocking the Raf/MEK/ERK-EIk-1 pathway with the ERK inhibitor PD98059 (Fig. 6). CRKL regulates erythroid and megakaryocyte differentiation of K562 cell via inactivating the Raf/MEK/ERK/Elk-1 pathway. However, CRKⅡ downregulation could not affect the Raf/MEK/ERK pathway (Fig. 7C, G), further indicating CRKⅡ has no effect on erythroid differentiation of K562 cells.
Taken together, we have illustrated for the first time that CRKL can inhibit erythroid and megakaryocyte differentiation of K562 cells via inactivating the Raf/MEK/ERK/Elk pathway. The novel action mechanism is outlined in Fig. 8. CRKL downregulation promotes the expression of Raf, p-Raf, p-MEK, p-ERK1/2 and EIk-1, then HMGB2 binds to the Gfi-1B promoter and enhances its transactivation by promoting the binding of Oct-1 and GATA-1 to the Gfi-1B promoter, which induces erythroid and megakaryocyte differentiation of K562 cells by increasing globin, hemoglobin and differentiation-specific genes expression. Taken together, we have established a new functional role and molecular pathway for CRKL during hematopoietic differentiation. These findings could be fundamental to the development of a novel potential diagnostic biomarker and therapeutic target for CML patients.