Prolactin-induced expression of cytokine-inducible SH2 signaling inhibitors in human hematopoietic progenitors.
OBJECTIVE
The prolactin (PRL) receptor (PRLR) utilizes the JAK2/STAT-5 pathway and induces expression of cytokine-inducible SH2 (CIS)/JAK2 binding (JAB) signaling inhibitors. We and others recently showed that CIS-3 and JAB abolish PRLR-mediated JAK2 activation and STAT-5 activity, whereas CIS-1, CIS-2, and CIS-4 had a negligible effect. Human CD34(+) hematopoietic progenitors express PRLRs and respond to PRL in vitro by enhanced cytokine-induced colony formation. To assess the signaling mechanism(s) involved in PRL-mediated enhancement of hematopoiesis and to identify further the CIS/JAB targets for PRL-mediated cellular responses, we assayed the effect of PRL, alone or in the presence of interleukin-3 (IL-3), on activation of STAT-5 and expression of CIS/JAB RNA in human cord blood (CB) CD34(+) cells.


METHODS
Isolated CB CD34(+) cells were incubated in serum-free cultures in the absence or presence of recombinant human (rh)PRL, rhIL-3, or both. Cell lysates were subjected to Western blot analysis with anti-STAT-5 and anti-phospho-STAT-5 antibodies. Isolated RNA was subjected to semiquantitative reverse transcriptase polymerase chain reaction analysis of CIS/JAB expression.


RESULTS
STAT-5 tyrosine phosphorylation was similarly induced by PRL and IL-3, with an additive effect detected in the presence of both stimuli. Both PRL and IL-3, alone or combined, failed to induce CIS-3 or JAB RNA expression in CD34(+) cells. Interferon-gamma had no effect on CIS-3/JAB induction in these cells. However, CIS-1 was induced by PRL < IL-3 < PRL+IL-3, whereas CIS-2 expression was induced by PRL = IL-3 < PRL+IL-3.


CONCLUSIONS
Our findings show that PRL induces activation of STAT-5 and expression of similar CIS/JAB family members as IL-3 does in human CB CD34(+) cells. Because CIS-1 abolishes STAT-5 activation via the IL-3 but not the PRL receptor, the hematopoietic growth-promoting effects of PRL may involve its capacity to provide sustained STAT-5-mediated stimulatory signals to the cells.
Cwikel
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Hematopoiesis Unit, Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion, Haifa, Israel.
Silvian-Drachsler
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Prolov
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Hooghe-Peters
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Merchav
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D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D007231:Infant, Newborn; D007377:Interleukin-3 / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D053614:Janus Kinase 2; D008894:Milk Proteins; D011388:Prolactin / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000071222:Suppressor of Cytokine Signaling 1 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D018909:src Homology Domains
D015703:Antigens, CD; D018952:Antigens, CD34; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D007377:Interleukin-3; D047908:Intracellular Signaling Peptides and Proteins; D053614:Janus Kinase 2; D008894:Milk Proteins; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012097:Repressor Proteins; D050799:STAT5 Transcription Factor; D000071222:Suppressor of Cytokine Signaling 1 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; C108944:SOCS1 protein, human; C507924:JAK2 protein, human