Effect of recombinant interleukin-6 and thrombopoietin on isolated guinea pig bone marrow megakaryocyte protein phosphorylation and proplatelet formation

Robert M. Leven, Barbara Clark, Fern Tablin

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12 Citations (Scopus)

Abstract

Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100 ng/ml IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 μg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was a potent stimulator of proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAKs and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.

Original languageEnglish (US)
Pages (from-to)252-268
Number of pages17
JournalBlood Cells, Molecules and Diseases
Volume23
Issue number2
DOIs
StatePublished - Aug 1997

Fingerprint

Thrombopoietin
Megakaryocytes
Interleukin-6
Guinea Pigs
Bone Marrow
Phosphorylation
Proteins
Phosphoric Monoester Hydrolases
Signal Transduction
Phosphotransferases
Collagen
Gels
Janus Kinases
Okadaic Acid
Phosphodiesterase Inhibitors
Protein-Tyrosine Kinases
Thymidine
Tyrosine
Cell Culture Techniques
Cytokines

Keywords

  • Growth factors
  • Megakaryocytopoiesis
  • Platelet formation
  • Signal transduction
  • Thrombopoietin

ASJC Scopus subject areas

  • Molecular Biology
  • Molecular Medicine
  • Hematology

Cite this

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title = "Effect of recombinant interleukin-6 and thrombopoietin on isolated guinea pig bone marrow megakaryocyte protein phosphorylation and proplatelet formation",
abstract = "Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6{\%} of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7{\%} at 100 ng/ml IL6 and 26.8{\%} at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 μg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was a potent stimulator of proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAKs and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.",
keywords = "Growth factors, Megakaryocytopoiesis, Platelet formation, Signal transduction, Thrombopoietin",
author = "Leven, {Robert M.} and Barbara Clark and Fern Tablin",
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T1 - Effect of recombinant interleukin-6 and thrombopoietin on isolated guinea pig bone marrow megakaryocyte protein phosphorylation and proplatelet formation

AU - Leven, Robert M.

AU - Clark, Barbara

AU - Tablin, Fern

PY - 1997/8

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N2 - Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100 ng/ml IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 μg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was a potent stimulator of proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAKs and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.

AB - Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100 ng/ml IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 μg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was a potent stimulator of proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAKs and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.

KW - Growth factors

KW - Megakaryocytopoiesis

KW - Platelet formation

KW - Signal transduction

KW - Thrombopoietin

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JO - Blood Cells, Molecules, and Diseases

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