Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells

Endothelial and endothelial progenitor cells (ECs and EPCs) play a fundamental role in angiogenesis that is essential for numerous physiological and pathological processes. The phosphatase and tensin homolog (PTEN)/ phosphoinositide 3-kinase (PI3K) pathway has been implicated in angiogenesis, but the mechanism in the regulation of this pathway in ECs and EPCs is poorly understood. Here we show that ARIA (apoptosis regulator through modulating IAP expression), a transmembrane protein that we recently identified, regulates the PTEN/PI3K pathway in ECs and EPCs and controls developmental and postnatal angiogenesis in vivo. We found that ARIA is abundantly expressed in EPCs and regulates their angiogenic functions by modulating PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling. Genetic deletion of ARIA caused nonfatal bleeding during embryogenesis, in association with increased small vessel density and altered expression of various vascular growth factors including angiopoietins and VEGF receptors. Postnatal neovascularization induced by critical limb ischemia was substantially enhanced in ARIA-null mice, in conjunction with more bone marrow (BM)-derived ECs detected in ischemic muscles. Administration of PI3K or NO synthase inhibitor completely abolished the enhanced neovascularization in ARIA(-/-) mice. Mechanistically, we identified that ARIA interacts with PTEN at the intracellular domain independently of the PTEN phosphorylation in its C-terminal tail. Overexpressed ARIA increased PTEN in the membrane fraction, whereas ARIA-silencing reduced the membrane-associated PTEN, resulting in modified PI3K/Akt signaling. Taken together, our findings establish a previously undescribed mode of regulation of the PTEN/PI3K/Akt pathway by ARIA, and reveal a unique mechanism in the control of angiogenesis. These functions of ARIA might offer a unique therapeutic potential.     

磷酸二酯酶抑制剂对乳鼠心肌细胞PI3K—Akt—eNOS信号通路的影响

目的：探讨西洛他唑（Cilostazol）对乳鼠心肌细胞PI3K-Akt-eNOS信号通路的影响。方法：观察西洛他唑对乳鼠心肌细胞NO影响的时效和量效关系，再用PI3K及eNOS抑制剂进行干预。检测NO的浓度，Western免疫印迹法检测总Akt、磷酸化Akt（p—Akt—ser473）及总eNOS、磷酸化eNOS（p-eNOS-Ser1177）表达水平。结果：西洛他唑升高心肌细胞NO浓度呈剂量和时间依赖性，不同浓度的西洛他唑均能升高Akt和eNOS磷酸化水平，但对Akt及eNOS总蛋白表达无明显影响。eNOS抑制剂L—NAME和P13K抑制剂Wortmannin均能抑制西洛他唑诱导的NO浓度升高，Wortmannin还能阻断西洛他唑诱导的Akt和eNOS的磷酸化。结论：西洛他唑可能激活乳鼠心肌细胞的PI3K—Akt—eNOS信号通路而促进NO的产生。     

Shear stress-induced activation of Tie2-dependent signaling pathway enhances reendothelialization capacity of early endothelial progenitor cells

Although endothelial progenitor cells (EPCs) play a pivotal role in the endothelial repair following arterial injury and shear stress has a beneficial effect on EPCs, however, the molecular mechanism underlying the influence of EPCs on the endothelial integrity and the regulation of shear stress on the EPC signaling remained to be studied. Here, we investigated the effects of laminar shear stress on the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)-dependent signaling and its relation to in vivo reendothelialization capacity of human early EPCs. The human early EPCs were treated with shear stress. Shear stress in a dose-dependent manner increased angiopoietin-2 (Ang2)-induced migratory, adhesive and proliferatory activities of EPCs. Transplantation of EPCs treated by shear stress facilitated in vivo reendothelialization in nude mouse model of carotid artery injury. In parallel, the phosphorylation of Tie2 and Akt of EPCs in response to shear stress was significantly enhanced. With treatment of Tie2 knockdown or Akt inhibition, shear stress-induced phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) of EPCs was markedly suppressed. After Tie2/PI3K/Akt/eNOS signaling was blocked, the effects of shear stress on in vitro function and in vivo reendothelialization capacity of EPCs were significantly inhibited. The present findings demonstrate for the first time that Tie2/PI3k/Akt/eNOS signaling pathway is, at least in part, involved in the EPCs-mediated reendothelialization after arterial injury. The upregulation of shear stress-induced Tie2-dependent signaling contributes to enhanced in vivo reendothelialization capacity of human EPCs.     

Reconstituted high-density lipoprotein inhibits thrombin-induced endothelial tissue factor expression through inhibition of RhoA and stimulation of phosphatidylinositol 3-kinase but not Akt/endothelial nitric oxide synthase

Endothelial cells express negligible amounts of tissue factor (TF) that can be induced by thrombin, which is important for acute coronary syndromes. Recent research suggests that endothelial TF expression is positively regulated by RhoA and p38mapk, but negatively by Akt/endothelial nitric oxide synthase (eNOS) pathway. High-density lipoprotein (HDL) is atheroprotective and exerts antiatherothrombotic effect. This study investigated the effect of a reconstituted HDL (rHDL) on endothelial TF expression induced by thrombin and the underlying mechanisms. In cultured human umbilical vein and aortic endothelial cells, thrombin (4 U/mL, 4 hours) increased TF protein level, which was reduced by rHDL (0.1 mg/mL, 43% inhibition, n=3 to 7, P<0.01). Activation of RhoA but not p38mapk by thrombin was prevented by rHDL. rHDL stimulated Akt/eNOS pathway. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 abolished the activation of Akt/eNOS and reversed the inhibitory effect of rHDL on TF expression. Adenoviral expression of the active PI3K mutant (p110) reduced TF expression stimulated by thrombin without inhibiting RhoA activation, whereas expression of the active Akt mutant (m/p) further facilitated TF upregulation by thrombin. Moreover, a dominant-negative Akt mutant (KA) reduced thrombin's effect and did not reverse the rHDL's inhibitory effect on TF expression. Inhibition of eNOS by N(omega)-nitro-L-arginine methyl ester (100 micromol/L) did not affect the rHDL's effect. In conclusion, rHDL inhibits thrombin-induced human endothelial TF expression through inhibition of RhoA and activation of PI3K but not Akt/eNOS. These findings implicate a novel mechanism of antiatherothrombotic effects of HDL.     

Downregulation of microRNA-126 in endothelial progenitor cells from diabetes patients, impairs their functional properties, via target gene Spred-1

Diabetes mellitus (DM) adversely affects the number and function of circulating endothelial progenitor cells (EPCs). Consequently, there is also a reduction in the repair mechanism of these cells, which is a critical and initiating factor in the development of diabetic vascular disease. The aim of the present study was to analyze miR expression profiles in EPCs from patients with DM and choose the most significantly regulated miR to study its possible role on EPC dysfunction and elucidate its mechanism of action. EPCs were collected from subjects with Type II DM and non-diabetic control subjects. Total RNA was harvested from EPCs, and a total of 5 candidate miRNAs were identified by microarray screening and were quantified by TaqMan real-time PCR. Lentiviral vectors expressing miR-126 and miR-126 inhibitor (anti-miR-126) were transfected into EPCs, and the EPC colony-forming capacity, proliferation activity, migratory activity, differentiation capacity, and apoptotic susceptibility were determined and Western Blotting and mRNA real-time PCR analyses were performed. To study the mechanisms, lentiviral vectors expressing Spred-1 and a short interfering RNA (siRNA) targeting Spred-1 were prepared. Five miRs were aberrantly downregulated in EPCs from DM patients. These miRs included miR-126, miR-21, miR-27a, miR-27b and miR-130a. Anti-miR-126 inhibited EPC proliferation, migration, and enhanced apoptosis. Restored miR-126 expression in EPCs from DM promoted EPC proliferation, migration, and inhibited EPC apoptosis ability. Despite this, miR-126 had no effect on EPC differentiation. miR-126 overexpression significantly downregulated Spred-1 in EPCs. The knockdown of Spred-1 expression in EPCs from DM promoted proliferation, migration, and inhibited apoptosis of the cells. The signal pathway of miR-126 effecting on EPCs is partially mediated through Ras/ERK/VEGF and PI3K/Akt/eNOS regulation. This study provides the first evidence that miR-126 is downregulated in EPCs from diabetic patients, and impairs EPCs-mediated function via its target, Spred-1, and through Ras/ERK/VEGF and PI3K/Akt/eNOS signal pathway.     

Activation of phosphatidylinositol 3-kinase is important for erythropoietin-induced erythropoiesis from CD34(+) hematopoietic progenitor cells

OBJECTIVE: Several transducing molecules, including JAK2, STAT5, MAP kinases, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma1, and PKC are activated by interaction between erythropoietin (EPO) and the EPO receptor. The aim of this was to examine the relative involvement of PI3K in the development of glycophorin A (GPA)(+) erythroid cells from normal hematopoietic progenitor cells. MATERIALS AND METHODS: CD34(+) hematopoietic progenitor cells or subpopulations obtained by FACS sorting were cultured in serum-free medium containing EPO with or without inhibitors for PI3K, p38, MEK, or PKC for various time periods before phenotypic analysis or detection of apoptosis by flow cytometry, cell cycle analysis, high-resolution tracking of cell division, Western blot analysis, or Akt kinase assay were performed. RESULTS: The PI3K inhibitor LY294002 completely counteracted the EPO-induced proliferation of CD34(+) progenitor cells and CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 also highly suppressed the expanded erythropoiesis induced by the combined action of EPO and stem cell factor. The profound inhibitory effect of LY294002 on proliferation was caused by its induction of cell cycle arrest in the G(0)/G(1) phase of the cell cycle. Some cells acquired GPA expression before they went through cell division. This was completely blocked by LY294002, implying an inhibitory effect on maturation. In addition, LY294002 completely blocked the viability-enhancing effect of EPO in CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 and various inhibitors of PKC completely suppressed the EPO-induced increase in the activity of Akt kinase, a direct downstream target of PI3K. CONCLUSIONS: Our results point to an important role for PI3K in mediating EPO-induced survival, proliferation, and possibly maturation of early erythroid progenitors.     

Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection

In the penis, nitric oxide (NO) can be formed by both neuronal NO synthase and endothelial NOS (eNOS). eNOS is activated by viscous drag/shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3kinase)/Akt pathway. Here we show that PI3-kinase/Akt physiologically mediates erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vasorelaxant drug papaverine cause rapid increases in phosphorylated (activated) Akt and eNOS. Phosphorylation is diminished by wortmannin and LY294002, inhibitors of PI3-kinase, the upstream activator of Akt. The two drugs also reduce erection. Penile erection elicited by papaverine is reduced profoundly in mice with targeted deletion of eNOS. Our findings support a model in which rapid, brief activation of neuronal NOS initiates the erectile process, whereas PI3-kinase/Akt-dependent phosphorylation and activation of eNOS leads to sustained NO production and maximal erection.     

Regulation of the RAP1/RAF-1/extracellularly regulated kinase-1/2 cascade and prolactin release by the phosphoinositide 3-kinase/AKT pathway in pituitary cells

In pituitary cells, prolactin (PRL) synthesis and release are controlled by multiple transduction pathways. In the GH4C1 somatolactotroph cell line, we previously reported that MAPK ERK-1/2 are a point of convergence between the pathways involved in the PRL gene regulation. In the present study, we focused on the involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the MAPK ERK-1/2 regulation and PRL secretion in pituitary cells. Either specific pharmacological PI3K and Akt inhibitors (LY294002, Akt I, and phosphoinositide analog-6) or Akt dominant-negative mutant (K179M) enhanced ERK-1/2 phosphorylation in unstimulated GH4C1 cells. Under the same conditions, PI3K and Akt inhibition also both increased Raf-1 kinase activity and the levels of GTP-bound (active form) monomeric G protein Rap1, which suggests that a down-regulation of the ERK-1/2 cascade is induced by the PI3K/Akt signaling pathway in unstimulated cells. On the contrary, ERK-1/2 phosphorylation, Raf-1 activity, and Rap1 activation were almost completely blocked in IGF-I-stimulated cells previously subjected to PI3K or Akt inhibition. Although the PRL promoter was not affected by either PI3K/Akt inhibition or activation, PRL release increased in response to the pharmacological PI3K/Akt inhibitors in unstimulated GH4C1 and rat pituitary primary cells. The IGF-I-stimulated PRL secretion was diminished, on the contrary, by the pharmacological PI3K/Akt inhibitors. Taken together, these findings indicate that the PI3K/Akt pathway exerts dual regulatory effects on both the Rap1/Raf-1/ERK-1/2 cascade and PRL release in pituitary cells, i.e. negative effects in unstimulated cells and positive ones in IGF-I-stimulated cells.     

DIDS对十字孢碱诱导心肌细胞凋亡中磷脂酰肌醇3激酶/蛋白激酶B信号转导的影响

目的探讨氯离子通道阻断剂DIDS对十字孢碱诱导心肌细胞凋亡与磷脂酰肌醇3激酶/蛋白激酶B信号及其下游分子一氧化氮合酶/一氧化氮的关系。方法实验分为对照组、十字孢碱组、DIDS组、LY294002(特异性磷脂酰肌醇3激酶抑制剂)组和L-NAME(非特异性一氧化氮合酶抑制剂)组。在十字孢碱诱导心肌细胞凋亡模型上,观察DIDS对心肌细胞存活率、凋亡和磷脂酰肌醇3激酶/蛋白激酶B及其下游分子一氧化氮合酶/一氧化氮的影响。结果与十字孢碱组比,DIDS明显改善了细胞存活率,提高了细胞磷酸化蛋白激酶B活性2.1倍(P<0.01),增加了一氧化氮合酶和磷酸化一氧化氮合酶的水平和一氧化氮水平(P<0.01);LY294002预处理完全抑制了磷酸化蛋白激酶B、一氧化氮合酶和磷酸化一氧化氮合酶水平的升高及升高的一氧化氮,完全阻断了DIDS的抗细胞凋亡作用;L-NAME预处理也使升高的一氧化氮水平下降,但仅部分阻断了DIDS的细胞保护作用。结论DIDS通过激活磷脂酰肌醇3激酶/蛋白激酶B信号通路发挥其抑制十字孢碱诱导的心肌细胞凋亡作用。     

Phosphatidylinositol-3,4,5-trisphosphate is required for insulin-like growth factor 1-mediated survival of 3T3-L1 preadipocytes

Adipocyte number, a determinant of adipose tissue mass, reflects the balance between the rates of proliferation/differentiation vs. apoptosis of preadipocytes. The percentage of 3T3-L1 preadipocytes undergoing cell death following serum deprivation was reduced by 10 nM insulin-like growth factor (IGF)-1 (from 50.0 +/- 0.7% for control starved cells to 27.5 +/- 3.1%). TUNEL staining confirmed the apoptotic nature of the cell death. The protective effect of IGF-1 was blocked by phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin, and LY294002, but was unaffected by rapamycin, PD98059, or SB203580, which inhibit mammalian target of rapamycin (mTOR), ERK kinase (MEK1), and p38 MAPK respectively. Exogenous PI(3,4,5)P3 (10 microM), the principal product of IGF-1-stimulated PI3K in 3T3-L1 preadipocytes, had a modest survival effect on its own, reducing cell death from 47.9 +/- 3.4% to 35.6 +/- 3.5%. When added to the combination of IGF-1 and LY294002, PI(3,4,5)P3 reversed most of the inhibitory effect of LY294002 on IGF-1-dependent cell survival, protein kinase B/Akt phosphorylation, and caspase-3 activity. Taken together, these results implicate PI(3,4,5)P3 as a necessary signal for the anti-apoptotic action of IGF-1 on 3T3-L1 preadipocytes.     

Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

SDF-1alpha/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow-derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1alpha (SDF-1alpha)-induced and stroma-induced chemotaxis and inhibited SDF-1alpha-induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1alpha/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor- and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.     

Activation of endothelial nitric oxide synthase by cilostazol via a cAMP/protein kinase A- and phosphatidylinositol 3-kinase/Akt-dependent mechanism

We investigated the effect of cilostazol on nitric oxide (NO) production in human aortic endothelial cells (HAEC). Cilostazol increased NO production in a concentration-dependent manner, and NO production was also increased by other cyclic-AMP (cAMP)-elevating agents (forskolin, cilostamide, and rolipram). Cilostazol increased intracellular cAMP level, and that effect was enhanced in the presence of forskolin. In Western blot analysis, cilostazol increased phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(1177) and of Akt at Ser(473) and dephosphorylation of eNOS at Thr(495). Cilostazol's regulation of eNOS phosphorylation was reversed by protein kinase A inhibitor peptide (PKAI) and by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Moreover, the cilostazol-induced increase in NO production was inhibited by PKAI, LY294002, and N(G)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor. In an in vitro model of angiogenesis, cilostazol-enhanced endothelial tube formation, an effect that was completely attenuated by inhibitors of PKA, PI3K, and NOS. These results suggest that cilostazol induces NO production by eNOS activation via a cAMP/PKA- and PI3K/Akt-dependent mechanism and that this effect is involved in capillary-like tube formation in HAEC.     

Dueling for dual inhibition: Means to enhance effectiveness of PI3K/Akt/mTOR inhibitors in AML

The phosphatidylinositol 3-kinase/protein kinase B (Akt)/mechanistic target of rapamycin (PI3K/Akt/mTOR) pathway is amplified in 60–80% of patients with acute myelogenous leukemia (AML). Since this complex pathway is crucial to cell functions such as growth, proliferation, and survival, inhibition of this pathway would be postulated to inhibit leukemia initiation and propagation. Inhibition of the mTORC1 pathway has met with limited success in AML due to multiple resistance mechanisms including direct insensitivity of the mTORC1 complex, feedback activation of the PI3k/Akt signaling network, insulin growth factor-1 (IGF-1) activation of PI3K, and others. This review explores the role of mTOR inhibition in AML, mechanisms of resistance, and means to improve outcomes through use of dual mTORC1/2 inhibitors or dual TORC/PI3K inhibitors. How these inhibitors interface with currently available therapies in AML will require additional preclinical experiments and conduct of well-designed clinical trials.     

Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax

The prevention of apoptosis is a key function of growth factors in the regulation of erythropoiesis. This study examined the role of the constitutively active serine/threonine kinase glycogen synthase kinase-3 (GSK3), a target of the phosphoinositide-3-kinase (PI3K)/Akt pathway, in the regulation of apoptosis in primary human erythroid progenitors. GSK3 phosphorylation at its key regulatory residues S21 (alpha isoform) and S9 (beta isoform) was high in steady-state culture, disappeared on growth factor withdrawal, and returned in response to treatment of cells with either erythropoietin or stem cell factor. Phosphorylation correlated with a PI3K-dependent reduction of 25% to 30% in measured GSK3 activity. LY294002, a specific inhibitor of PI3K, induced apoptosis in growth factor-replete erythroid cells to a degree similar to growth factor deprivation, whereas the Mek1 inhibitor U0126 had no effect, implicating PI3K and not mitogen-activated protein kinase in survival signaling. Growth factor-deprived erythroblasts, which undergo apoptosis rapidly, were protected from apoptosis by both lithium chloride, a GSK3 selective inhibitor, and inhibition of caspase activity. However, the clonogenic potential of single cells, which more accurately reflects cell survival, was maintained by lithium chloride, but not by caspase inhibition. Furthermore, lithium chloride, but not caspase inhibition, prevented the appearance of the conformational form of Bax associated with apoptosis induction. In summary, GSK3 activity is suppressed by erythropoietin and stem cell factor in human erythroid progenitor cells, and increased GSK3 activity, brought about by growth factor withdrawal, may regulate commitment to cell death through a caspase-independent pathway that results in a conformational change in Bax.     

Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death

OBJECTIVE: The signaling pathways mediating proliferation and apoptosis in vascular smooth muscle cells (VSMC) are not well established. It has previously been shown that activation of the phosphoinositide 3-OH kinase (PI3K)/Akt pathway or the ERK 1/2 pathway can mediate anti-apoptotic function in different cell types. This study determined the specific contribution of the PI3K/Akt and ERK pathway in the regulation of apoptosis and proliferation of VSMC. METHODS AND RESULTS: Incubation of rat VSMC with FCS, insulin or IGF-1 time-dependently stimulated the phosphorylation of Akt, however FCS but not insulin or IGF-1 activated the MAP-kinase ERK 1/2. Moreover, insulin inhibited H(2)O(2)-induced apoptosis via the Akt pathway as demonstrated by pharmacological inhibition of the PI3K or overexpression of a dominant negative Akt mutant. In contrast, FCS inhibited H(2)O(2)-induced apoptosis via the Akt and also the ERK pathway. FCS, but not insulin or IGF-1 induced VSMC proliferation, suggesting that Akt activation is necessary but not sufficient for VSMC proliferation. FCS-induced proliferation of VSMC was only mediated via the Akt pathway and not the ERK pathway. CONCLUSIONS: These results define a link between cell proliferation and programmed cell death in VSMC via the same signal transduction pathway, namely activation of the serine/threonine kinase Akt, which may have significant implication for the development of vascular diseases or remodeling.     

PI3K/Akt/mTOR信号转导通路在肝细胞癌发生发展中的作用

PI3K/Akt/mTOR信号转导通路可以通过调控基因表达,在肝细胞癌肿瘤细胞生成、增殖、转移和凋亡等过程中发挥重要作用。简述了PI3K/Akt/mTOR信号通路的组成及功能,对PI3K/Akt/mTOR通路在肝细胞癌进程中的作用机制及相关抑制剂的研究进展进行了综述,揭示阻断PI3K/Akt/mTOR通路可以成为肝细胞癌治疗新的靶点方向。     

Stromal cell-derived factor-1alpha stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C

The stromal cell-derived factor-1 (SDF-1) is an alpha chemokine that binds to the CXCR4 receptor. Knock-out studies in mice demonstrate that this ligand-receptor pair is essential in hematopoiesis. One function of SDF-1 appears to be the regulation of migration of hematopoietic progenitor cells. We previously characterized signal transduction pathways induced by SDF-1alpha in human hematopoietic progenitors and found tyrosine phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK), the adaptor molecule p130 Cas, and the cytoskeletal protein paxillin. To better understand the functional role of signaling molecules connecting the CXCR4 receptor to the process of hematopoietic migration, we studied SDF-1alpha-mediated pathways in a model hematopoietic progenitor cell line (CTS), as well as in primary human bone marrow CD34(+) cells. We observed that several other focal adhesion components, including focal adhesion kinase (FAK) and the adaptor molecules Crk and Crk-L, are phosphorylated on SDF-1alpha stimulation. Using a series of specific small molecule inhibitors, both protein kinase C (PKC) and phosphoinositide-3 kinase (PI-3K) appeared to be required for SDF-1alpha-mediated phosphorylation of focal adhesion proteins and the migration of both CTS and primary marrow CD34(+) cells, whereas the mitogen-activated protein kinases ERK-1 and -2 were not. These studies further delineate the molecular pathways mediating hematopoietic progenitor migration and response to an essential chemokine, SDF-1alpha. (Blood. 2000;95:2505-2513)