Autocrine Growth Factor Production Research Articles

Role of Src in Signal Transduction Pathways

Src was the first oncogene to be discovered, and the first protein tyrosine kinase. The study of how Src transforms cells has been a rich field that has lead to insights into the control of the cell cycle, the organization of the cytoskeleton, and growth factor-independent growth. Yet we still do not fully understand exactly what Src does. In normal cells, Src has been implicated in the control of cell division, the production of autocrine growth factors, the cell's survival response, as well as in cell motility. My laboratory has focused on the involvement of Src and related kinases in the response of cells to mitogenic growth factors. We have shown that the activity of Src kinases is necessary for cells to enter the cell cycle when treated with mitogens such as platelet-derived growth factor. Src activity initiates a signal transduction cascade, involving the adaptor protein She, which culminates in the transcriptional activation of the transcription factor Myc. Furthermore, we have also shown that this requirement for Src is abrogated in cells lacking the tumour suppressor p53, suggesting that another of Src's functions in normal cells is to suppress the actions of p53.

Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts.

In the present study, we examined the underlying mechanism, which causes the constitutive tyrosine phosphorylation of signal transducer and activator of transcription 5 (STAT5) in acute myeloid leukemia (AML) blasts. Constitutive STAT5 phosphorylation was observed in 18 of 26 (69%) patients with AML. The constitutive STAT5 phosphorylation was caused by different mechanisms. In the majority of the investigated cases (71% (12 of 17)) constitutive STAT5 phosphorylation was associated with autophosphorylation of the type III receptor tyrosine kinase Flt3. In 47% (eight of 17) of these cases autophosphorylation of Flt3 coincided with tandem duplications of the Flt3 gene, resulting in constitutive phosphorylation of the receptor, while 24% (four of 17) of the cases demonstrated STAT5 phosphorylation and Flt3 autophosphorylation without mutations. In addition, a subset of AML cases (29% (five of 17)) had no autophosphorylation of the Flt3 receptor, but demonstrated constitutive STAT5 phosphorylation, which was partly due to autocrine growth factor production. All AML cases with high STAT5 and Flt3 phosphorylation demonstrated, in general, a lower percentage of spontaneous apoptosis, compared to AML blasts with no spontaneous STAT5 phosphorylation. Addition of the receptor tyrosine III kinase inhibitor AG1296 strongly inhibited STAT5 phosphorylation and enhanced the percentage of apoptotic cells without modulating the Bcl-xl protein levels. These data indicate that in the majority of AML cases the constitutive STAT5 phosphorylation is caused by Flt3 phosphorylation mostly due to mutations in the receptors and associated with a low degree of spontaneous apoptosis.

Role of Src in Signal Transduction Pathways

Src was the first oncogene to be discovered, and the first protein tyrosine kinase. The study of how Src transforms cells has been a rich field that has lead to insights into the control of the cell cycle, the organization of the cytoskeleton, and growth factor-independent growth. Yet we still do not fully understand exactly what Src does. In normal cells, Src has been implicated in the control of cell division, the production of autocrine growth factors, the cell's survival response, as well as in cell motility. My laboratory has focused on the involvement of Src and related kinases in the response of cells to mitogenic growth factors. We have shown that the activity of Src kinases is necessary for cells to enter the cell cycle when treated with mitogens such as platelet-derived growth factor. Src activity initiates a signal transduction cascade, involving the adaptor protein She, which culminates in the transcriptional activation of the transcription factor Myc. Furthermore, we have also shown that this requirement for Src is abrogated in cells lacking the tumour suppressor p53, suggesting that another of Src's functions in normal cells is to suppress the actions of p53.

Leukemic cells from murine myeloid leukemia display an intrinsic ability for autonomous proliferation

Objective. Human acute myeloid leukemia (AML) cells can proliferate in vitro in the absence of added growth factors when cultured at high cell density. Autocrine growth factor production is a postulated mechanism of autonomous growth. We sought to examine this using murine AML cells. Materials and Methods. We have utilized a Moloney murine leukemia virus (M-MuLV) model of AML to investigate the nature of autonomous in vitro growth of myeloid leukemic cells. Results. Like human AML, M-MuLV-induced myeloid leukemic cells displayed autonomous growth in unstimulated high cell density cultures. However, replating of individual, primary, growth factor autonomous colonies of leukemic cells demonstrated the presence of clonogenic cells capable of autonomous growth when cultured at low cell density. In addition, there was heterogeneity in the progeny of these cells: both factor-dependent leukemic cells and cells autonomous of exogenous factor were observed. Conclusion. We propose that clonogenic cells capable of autonomous growth at low cell density represent leukemic progenitors while the majority of leukemic cells derived from these “autonomous” leukemic cells are factor-dependent.

Disruption of cadherin-related junctions triggers autocrine expression of vascular endothelial growth factor in bovine aortic endothelial cells : effects on cell proliferation and death resistance.

The mechanisms involved in the blockade of proliferation in confluent endothelial cells are insufficiently understood. In this regard, the continuity of intercellular junctions appears to be critical to the regulation of endothelial monolayer cell growth. The present study examined the hypothesis that the disruption of the intercellular adherens junctions will trigger both endothelial cell proliferation and autocrine production of growth factors. With this purpose, we assessed the changes in growth, death resistance, and expression of vascular endothelial growth factor (VEGF) under conditions of disruption of the intercellular junctions between endothelial cells. Disruption of cell junctions was produced by means of a specific anti-vascular endothelial cadherin monoclonal antibody, EGTA, or cytochalasin D. Our results disclosed that these maneuvers induce an increase in VEGF mRNA production, with transcription of the 121-, 165-, and 189-amino acid isoforms of VEGF. Further evidence of the relationship between endothelial cells monolayer continuity and VEGF protein expression was obtained by the demonstration of an increase in VEGF protein, as determined by Western blot, induced by the aforementioned maneuvers, as well as by immunocytochemical detection of increased VEGF staining in the areas surrounding a mechanical endothelial injury and in endothelial cells at subconfluence. In functional terms, the autocrine expression of VEGF was associated with growth-promoting and cytoprotective effects, as assessed by [(3)H]thymidine uptake, (51)Cr release, and flow cytometry. In conclusion, our results reveal that disruption of homophilic interendothelial junctions induces VEGF expression. Under these conditions, autocrine VEGF appears to have a relevant role in death inhibition and proliferation of endothelial cells.

TGFbeta isoforms and decorin gene expression are modified in fibroblasts obtained from non-syndromic cleft lip and palate subjects.

Interaction between extracellular matrix (ECM) and cytokines is thought to be crucial for palatal development. The localization of transforming growth factors (TGFalpha and TGFbeta isoforms) in craniofacial tissues suggests that they carry out multiple functions during development. In the present report, we studied TGFalpha, TGFbeta1, and TGFbeta3 expressions and their effects on ECM macromolecule production of normal and cleft palatal fibroblasts in vitro, to investigate the mechanisms by which the phenotypic modulation of fibroblasts occurs during the cleft palate process. The results indicated that, while TGFalpha mRNA was not evidenced in CLP or normal fibroblasts, a reduced TGFbeta1 hybridization signal was detected in CLP fibroblasts. In addition, these secreted more active TGFbeta3 than TGFbeta1, as evaluated in a biological assay. The CLP phenotype, which differed from the normal one because of its higher PG decorin expression and greater production of GAG and collagen, was further modified by the addition of growth factors. In fact, in CLP fibroblasts, TGFalpha and TGFbeta1 down-regulated PG decorin transcript, TGFbeta1 increased collagen and GAG in both cellular and extracellular compartments, and TGFbeta3 promoted secretory processes of cells. In conclusion, the data represent the first report in a human model in vitro that TGFbeta1 and beta3 are differently expressed and are correlated to the CLP phenotype. Thus, strength is given to the hypothesis that TGFbeta isoforms are the potential inducers of phenotypic expression in palatal fibroblasts during development and that an autocrine growth factor production mechanism may be responsible for the phenotypic modifications.

Melanomas, from the cell cycle point of view (Review).

Transformation of melanocytes to metastatic melanoma cells is characterized by unrestricted proliferation under growth-factor-deprived conditions, genetic loss of cyclin dependent kinase (CDK) inhibitors (CKI, e.g. p16INK4A), and aberrant production of autocrine growth factors (e.g. basic fibroblast growth factor). The latter induces increased expression of positive CDK regulators (e.g. cyclin D1) and reduced expression of additional CKIs (e.g. p27KIP1). Combined, these events lead to sustained CDK activity and hyperphosphorylation/inactivation of the retinoblastoma tumor suppressor protein (Rb). The persistent Rb phosphorylation causes the accumulation of E2F and the transcription of its target genes whose products promote cell cycle progression.

Protein kinase C-ε associates with the Raf-1 kinase and induces the production of growth factors that stimulate Raf-1 activity

Several observations indicate that the Raf-1 kinase is a downstream effector of protein kinase C-epsilon (PKC epsilon). We recently have shown that Raf-1 is constitutively activated in PKC epsilon transformed Rat6 fibroblasts, and transformation can be reverted by expression of a dominant negative Raf-1, but not a dominant negative Ras mutant (Cacace et al., 1996). Cai et al. (1997) demonstrated that PKC epsilon induced proliferation of NIH3T3 cells is independent of Ras or Src, but depends on Raf-1. These authors further suggested that PKC epsilon activates Raf-1 by direct phosphorylation. Here we have investigated the functional interaction between PKC epsilon and Raf-1. PKC epsilon, but not PKC alpha, was found to bind to the Raf-1 kinase domain. The association appeared to be direct, as it could be reconstituted in vitro with purified proteins. Raf-1 and PKC epsilon could be co-precipitated from Sf-9 insect cells and PKC epsilon transformed NIH313 cells (NIH/epsilon). The association was negatively regulated by ATP in vitro and by TPA treatment in NIH/epsilon cells, but not in Sf-9 insect cells. Raf-1 was constitutively activated in NIH/epsilon cells. However, using coexpression experiments in Sf-9 cells and transiently transfected A293 cells we did not obtain any evidence for a direct activation of Raf-1 by PKC epsilon. PKC epsilon did not induce translocation of Raf-1 to the membrane. Furthermore, PKC epsilon did not activate Raf-1 nor enhance the kinase activity of Raf-1 that had been pre-activated by coexpression of Ras or the Lck tyrosine kinase. In contrast, conditioned media from PKC epsilon transformed cells induced a robust activation of Raf-1. This activation could be partially reproduced by recombinant TGFbeta, a growth factors secreted by PKC epsilon transformed Rat6 cells. In conclusion, our results suggest that PKC epsilon stimulates Raf-1 indirectly by inducing the production of autocrine growth factors.

Delayed eosinophil programmed cell death in vitro: A common feature of inhalant allergy and extrinsic and intrinsic atopic dermatitis

The present studies were undertaken to characterize the potential role of eosinophil programmed cell death (PCD) in atopic diseases. Peripheral blood eosinophil PCD was found to be delayed in inhalant allergy ( p < 0.05) and delayed to an even greater extent in atopic dermatitis (AD) ( p < 0.0001) when compared to nonatopic subjects. There was no difference in the occurrence of PCD between the extrinsic and the intrinsic type of AD, pointing to a secondary role of specific sensitization. Blockade of eosinophil PCD was not responsible for peripheral blood eosinophilia, because we found no obvious relationship of eosinophil survival to blood eosinophil count. Eosinophil supernatants of more patients with AD than of patients with inhalant allergy dose-dependently inhibited PCD in nonatopic eosinophils, and it was shown that this effect was possibly due to autocrine production of granulocyte-macrophage–colony stimulating factor, probably IL-5. Eosinophil expression of CD95 (Fas antigen) did not change over time in culture and was not modulated by cytokines prolonging eosinophil survival. In contrast, IL-3, IL-5, and granulocyte-macrophage–colony stimulating factor caused an upregulated expression of CD69. However, in AD, CD69 on eosinophils was upregulated without the need of exogenous growth factor or factors over time in culture, thus confirming an autocrine production of proeosinophilic cytokines. In conclusion, our data clearly indicate that eosinophil PCD is markedly delayed in the so-called atopic diseases irrespective of allergen sensitization and suggest that this effect is mediated by the autocrine production of growth factors by eosinophils. (J Allergy Clin Immunol 1997;100:536-43.)

Interleukin-15 Stimulates C2 Skeletal Myoblast Differentiation

Interleukin-15 (IL-15) is a cytokine which is highly expressed in skeletal muscle, and which stimulates muscle protein accretion in cultured skeletal muscle fibers. Using parental C2 skeletal myoblasts, no significant effects of IL-15 on skeletal muscle differentiation were observed. To test the hypothesis that IL-15 may stimulate skeletal muscle differentiation if the strong differentiation-inducing effects of autocrine insulin-like growth factor (IGF) production were inhibited, a C2 myoblast subline (C2-pBP4) was stably transfected with an expression vector for rat IGF binding protein-4 (IGFBP-4). Differentiation responses to autocrine and exogenous IGFs in C2-BP4 myoblasts were reduced 3- to 4-fold in C2-BP4 cultures compared to C2-pLXSN cultures, a subline transfected with a control plasmid. Addition of IL-15 to C2-pBP4 myoblasts doubled the number of differentiated muscle cells which arose. These findings indicate that IL-15 can stimulate myogenic differentiation in conditions in which the strongly differentiative effects of the IGFs are inhibited. The differentiative activity of IL-15 may be of physiological significance in conditions in which IGF concentrations are low or in which the IGFs are sequestered by binding proteins.

Hormonal regulation of uterine macrophages.

Macrophages are major cellular inhabitants of cycling and pregnant mammalian uteri. Their densities and patterns of tissue distribution in this organ fluctuate in concert with levels of circulating female sex steroid hormones, estrogens and progesterone, and their production of various effector molecules also may be hormonally regulated. Hormonal control may be achieved by direct binding to receptors or by indirect pathways where hormones modulate production of various autocrine and paracrine cytokines and growth factors that then target to resident macrophages and influence their secretory profiles. In this paper, we marshall evidence supporting the concept that progesterone acts as a powerful negative regulator of these versatile cells, reducing their migration into the uterus and impairing their ability to produce potent effector molecules such as nitric oxide that could interfere with the success of pregnancy.

Glucosamine for psoriasis?

Amphiregulin and transforming growth factor-α, agonists for the epidermal growth factor receptor, are the major autocrine growth factors for cultured keratinocytes, and their substantial overexpression in psoriatic lesions suggests that they are crucial to the basal hyperplasia that characterizes psoriasis. Amphiregulin binds to heparin and related highly sulfated polysaccharides, and exogenous heparin blocks its growth factor activity, rationalizing previous reports that psoriasis responds to heparin therapy. Differentiating keratinocytes produce increased amounts of protein-bound as well as free-chain heparan sulfates, which may function physiologically as amphiregulin antagonists. By promoting keratinocyte synthesis of these heparan sulfates, glucosamine administration may inhibit amphiregulin function and thus provide therapeutic benefit in psoriasis. Concurrent ingestion of fish oil, by impeding the excessive activation of protein kinase C, may decrease keratinocyte production of amphiregulin and other autocrine growth factors, thus complementing the postulated benefits of glucosamine.

Bcl-2 Expression in Acute Myeloblastic Leukaemia: Relationship with Autonomous Growth and CD34 Antigen Expression

The bcl-2 gene encodes a mitochondrial protein that inhibits the onset of apoptosis induced by growth factor withdrawal or cytotoxic agents. Using quantitative flow cytometry and expressing bcl-2 levels as the number of molecules of equivalent soluble fluorochrome (MESF) per cell, we have shown that bcl-2 protein expression in the blast cells from patients with acute myeloblastic leukaemia (AML) is heterogeneous, but not related to FAB type. The blast cells from AML patients with the capacity to grow and survive autonomously in vitro were found to have higher bcl-2 MESF values than those that were dependent upon exogenous growth factors. We have previously reported that the blast cells from 70% of AML patients exhibit autonomous growth and autocrine growth factor production in vitro and that this has been shown to be an important indicator of poor prognosis in AML. High bcl-2 expression has also been associated with a low complete remission rate and poor survival in AML.In the patients whose blast cells exhibited autonomous growth, neutralisation of endogenous GM-CSF resulted in down-regulation of bcl-2 protein, whereas in blast cells from patients whose cells proliferated only in the presence of added growth factors, incorporation of recombinant human (rh) GM-CSF in the culture media resulted in up-regulation of bcl-2. Because CD34 positivity has been reported as another indicator of poor prognosis in AML, we compared bcl-2 expression in cases of CD34 positive AML, CD34 negative AML and CD34 positive normal bone marrow cells. Bcl-2 was found to be strongly expressed on the CD34+ normal bone marrow cells. The blast celb from CD34+ AML patients expressed significantly higher bcl-2 levels than CD34- AML patients. In five cases of CD34+ AML, the bcl-2 levels were determined on purified CD34+ and CD34- blast cell populations. The CD34+ blast cells were found to express significantly higher bcl-2 levels compared with the CD34- blast cells. Our data would suggest that quantification of bcl-2 in AML blast cells may be useful as a prognostic indicator in AML.

Modelling the consequences of interactions between tumour cells.

Classical models of tumorigenesis assume that the mutations which cause tumours to grow act in a cell-autonomous fashion. This is not necessarily true. Sometimes tumour cells may adopt genetic strategies that boost their own replication and which also influence other cells in the tumour, whether directly or as a side-effect. Tumour growth as a whole might be enhanced or retarded. We have used mathematical models to study two non-autonomous strategies that tumour cells may use. First, we have considered the production by tumour cells of an angiogenesis growth factor that benefits both the cell from which it originates and neighbouring cells. Second, we have analysed a situation in which tumour cells produce autocrine-only or paracrine-only growth factors to prevent programmed cell death. In the angiogenesis model, stable genetic polymorphisms are likely to occur between cells producing and not producing the growth factor. In the programmed cell death model, cells with autocrine growth factor production can spread throughout the tumour. Production of paracrine-only growth factor is never selected because it is 'altruistic' (that is of no benefit to the cell that makes the growth factor), despite being potentially beneficial to tumour growth as a whole. No polymorphisms can occur in the programmed cell death model. Production of angiogenesis and other growth factors in tumours may be under stable genetic, rather than epigenetic, control, with implications for therapies aimed at such targets. Many of the mutations observed in tumours may have non-autonomous effects.

Antitumor activity of combined blockade of epidermal growth factor receptor and protein kinase A.

Epidermal growth factor (EGF)-related proteins, such as transforming growth factor-alpha (TGF-alpha), control cancer cell growth through hormonal pathways (i.e., autocrine [hormone acts on cell that produces it] and paracrine [hormone acts on nearby cells] pathways). Overexpression of TGF-alpha and/or its receptor (EGFR) has been detected in human cancers. The blockade of EGFR activation by the use of anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is generally overexpressed in human cancer cells and is involved in neoplastic transformation. Inhibition of PKAI by selective cAMP analogues, such as 8-chloro-cAMP (8-CI-cAMP), induces growth inhibition in various human cancer cell lines. On the basis of our previous observations of a cooperative anti-proliferative effect of anti-EGFR Mab 528 and 8-Cl-cAMP in human cancer cell lines in vitro, we evaluated the anticancer activity in vivo of the combination of an anti-EGFR MAb (MAb C225) and 8-Cl-cAMP. Athymic mice were injected subcutaneously with 10(7) human colon carcinoma GEO cells. After 7 days, when established tumor xenografts of 0.30-0.35 cm3 were detectable, 10-15 mice per group were treated intraperitoneally twice weekly with different doses of 8-Cl-cAMP and/or MAb C225. Cancer cell expression of various growth factors was evaluated by immunohistochemical analysis in tumors obtained from control and treated mice. Data were evaluated for statistical significance using the Student's t test and the Mantel-Cox logrank test. All P values represent two-sided tests of statistical significance. A 5-week treatment with low doses of 8-Cl-cAMP (0.5 mg/dose) and MAb C225 (0.25 mg/dose) blocked GEO tumor growth (compared with that in control mice; P < .00001) and suppressed cancer cell production of autocrine growth factors, such as TGF-alpha, amphiregulin, and CRIPTO, and of angiogenic (promotes new blood vessel formation) factors, such as vascular endothelial growth factor and basic fibroblast growth factor, with no signs of toxicity. Control and 8-Cl-cAMP (0.5 mg/dose)-treated mice died within 9-10 weeks after tumor cell injection. In MAb C225 (0.25 mg/dose)-treated mice, GEO tumors resumed a growth rate comparable to that in control animals within 3 weeks following the end of treatment and the mice died between 11 and 20 weeks after tumor cell injection. GEO tumor growth was significantly delayed in the MAb C225 plus 8-Cl-cAMP treatment group (P < .00001) and was accompanied by a prolonged survival of mice (P < .00001) as compared with the control group. Long-term treatment with a combination of agents that selectively inhibit two intracellular signal-transduction enzymes, such as the PKAI serine-threonine kinase and the EGFR tyrosine kinase, has anticancer activity in vivo, reflected by suppression of tumor proliferation and angiogenesis, with no signs of toxicity. Since these inhibitors of intracellular mitogenic (growth-stimulating) signaling have a different mechanism(s) of action and do not antagonize the effects of cytotoxic therapy, a combination of anti-EGFR MAb C225 and 8-Cl-cAMP should be investigated as a nontoxic, long-term treatment for cancer patients following chemotherapy.

Hormonal control of growth factor receptor expression.

In this report, we have discussed a series of results obtained in our laboratory that, together with data by other authors, demonstrate that the expression of the erbB-2 tyrosine kinase receptor oncogene in breast cancer cells is regulated by multiple factors and hormones, which modulate their growth and differentiation. In particular, we have shown that estrogens specifically inhibit erbB-2 expression by transcriptional repression, which is exerted through a sequence within the erbB-2 gene promoter. Estrogens control mammary cell growth directly, by inducing early gene expression, and indirectly, by increasing autocrine growth factor production or decreasing growth inhibitors. The data presented here suggest that mammary cells respond to estrogen also by modifying the receptor array on their surface, thus setting their own sensitivity to the different autocrine and paracrine factors. As a first consequence, the modulation of erbB-2 expression level by antiestrogen may represent a point to consider when selecting breast cancer patients for hormonal therapy, in those (few) cases where estrogen receptor positivity accompanies erbB-2 amplification. On the other hand, antiestrogen-induced upregulation of erbB-2 may improve tumor targeting of drugs designed to interact or interfere with erbB-2, such as humanized antibodies, immunotoxins, or engineered ligands. These possibilities should be tested in appropriate model systems in the future.

Analysis of oncogenes, tumor suppressor genes, autocrine growth‐factor production, and differentiation state of human osteosarcoma cell lines

Human osteosarcoma and fibrosarcoma cell lines were investigated for alterations in oncogenes, tumor suppressor genes, and growth factors, all of which have been implicated in tumor formation. Characterization of oncogenes that are involved in osteosarcoma formation, including the c-fos and c-myc oncogenes, indicated that all six osteosarcoma cell lines examined had 5- to 20-fold amplification of the c-myc oncogene, whereas neither of two fibrosarcoma cell lines c-myc amplification. Interestingly, only three of six osteosarcoma cell lines displayed altered c-myc immediate-early gene function. c-fos was found to be normal, both at the gene and functional levels, in all six osteosarcoma and both fibrosarcoma cell lines tested. Characterization of two tumor suppressor genes, p53 and RB1, that have been implicated in osteosarcoma formation indicated that p53 was altered in five of six osteosarcoma cell lines, whereas RB1 was altered in only two or six of these cell lines. Neither RB1 nor p53 was found to be altered in the fibrosarcoma cell lines tested. An additional transformation marker, autocrine growth-factor production, was observed in all six osteosarcoma cell lines and both fibrosarcoma cell lines examined. Finally, the differentiation state of the osteosarcoma cell lines was investigated via the bone differentiation markers alkaline phosphates and osteocalcin. Alkaline phosphatase activity was observed in four of six osteosarcoma cell lines but not in the two fibrosarcoma cell lines examined. The alkaline phosphatase activity was a result of the expression of the bone/liver/kidney alkaline phosphatase isoform. High-level osteocalcin expression was observed in one of the osteosarcoma cell lines but not in the two fibrosarcoma cell lines examined, although all cell lines demonstrated low-level osteocalcin expression. Together, these data demonstrate that relatively undifferentiated osteosarcomas commonly display c-myc amplification, p53 and RB1 mutation, and autocrine growth-factor production, all of which may play a role in osteosarcomagenesis.

Involvement of interleukin 1 and tumour necrosis factor alpha as endogenous growth factors in human osteoblastic cells.

Normal human osteoblastic (OB) cells produce several haematopoietic factors, including IL-1, IL-6, PGE2 and TNF-alpha. However, it is unknown whether these factors play a role as autocrine mitogenic factors. We show here that some of these cytokines released by OB cells are endogenous growth factors for OB cells. Conditioned medium (CM) obtained from quiescent OB cells, dose-dependently stimulated OB cell proliferation, suggesting the production of autocrine growth factors by OB cells. Treatment with exogenous rhIL-1 and rhTNF-alpha increased OB cell growth. We found that neutralizing antibodies against IL-1 and TNF-alpha at concentrations that specifically inhibited the mitogenic activity of these cytokines, suppressed part of the mitogenic effect of CM on quiescent OB cells cultured at low or high density. In contrast, treatment of OB cells with indomethacin at a dose (10(-6) M) that inhibits endogenous prostaglandin production, increased OB cell proliferation in the presence or absence of CM, indicating that the mitogenic effect of CM on OB cells was not due to PGE2. In addition, exogenous recombinant human (rh)IL-6, or a specific neutralizing anti-IL-6 antibody, did not affect the OB cell proliferation. The results indicate that, in contrast to PGE2 and IL-6, IL-1 and TNF-alpha released by OB cells act as endogenous mitogenic factors for human osteoblasts.

Expression and Physiologic Significance of Kit Ligand and Stem Cell Tyrosine Kinase-1 Receptor Ligand in Normal Human CD34+, c-Kit+ Marrow Cells

To determine the potential role of autocrine growth factor production in regulating primitive human hematopoietic cell development, we examined highly purified CD34+, c-Kit+ marrow mononuclear cells for expression of c-Kit ligand (KL) and stem cell tyrosine kinase 1 (stk1) ligand (STK1-L). Normal marrow mononuclear cells coexpressing CD34 and c-Kit were isolated by a combination of immunomagnetic bead isolation and fluorescence-activated cell sorting. Purified cells were then screened for expression of KL and stk1-L mRNA using a sensitive reverse transcription-polymerase chain reaction method. Using this approach, expression of both cytokine genes at the mRNA level was found in this highly enriched cell population. We then examined the functional significance of these mRNAs by inhibiting their expression with antisense (AS) oligodeoxynucleotides (ODN). In comparison to untreated or control ODN treated cells, inhibition of KL led to a 70% and 89% inhibition in burst-forming unit-erythroid (BFU-E) and colony-forming unit-Mix (CFU-Mix) colonies but had no significant effect on CFU-granulocyte-macrophage (CFU-GM) cloning efficiency. In contrast, inhibition of STK1-L alone had no effect on colony formation. However, when STK1-L AS ODN was combined with KL AS ODN, additive inhibition of CFU-GM and CFU-MIX but not of BFU-E colonies was observed. These findings, along with those of our previous studies showing inhibition of primitive hematopoietic cell growth with antisense ODN directed towards the stk1 receptor, suggest the possibility that both receptor/ligand axes regulate primitive hematopoietic cell growth via an autocrine growth loop.

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1.

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-alpha) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-alpha and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth of IGF-1 requires activation of an EGF receptor-mediated autocrine loop.