Differential expression and phosphorylation of distinct STAT3 proteins during granulocytic differentiation

Abstract

External stimuli act in concert with intracellular signals to regulate a cell's genetic program, activating genes important in granulocytic lineage commitment, proliferation, and maturation. Signal transducer and activator of transcription 3 (STAT3), a transcription factor, has been implicated in mediating granulocytic differentiation. We have examined the role of STAT3 as a physiologic mediator of granulocytic kinetics. Distinct isoformsthe long form STAT3␣, the truncated forms STAT3␤ and STAT3␥, and a putative novel form STAT3␦-were expressed and activated in a maturation stage-specific manner. With the progression of differentiation, the ratio of isoforms shifted from predominantly STAT3␣ to STAT3␤. The kinetics of STAT3␥, generated through proteolytic cleavage of STAT3␣, coincided with but were inverse to those of STAT3␣. STAT3␦ was expressed at low levels and decreased with differentiation but was preferentially phosphorylated during an intermediate IntroductionHematopoietic lineage determination by transcriptional collaboration with cytokines is well established. 1 In particular, members of the STAT (signal transducer and activator of transcription) transcription factor family mediate many cytokine-induced responses in hematopoietic cells, including proliferation, differentiation, and survival. 2 Upon ligand binding, STATs are recruited to activated cell-surface receptors and become phosphorylated on a specific residue either directly or through association with a Janus kinase. Subsequently, the phosphorylated STATs homodimerize or heterodimerize and translocate to the nucleus, where they regulate transcription by binding to specific DNA promoter elements. 2,3 The STAT family consists of 7 members, most of which are ubiquitously expressed. However, individual STAT proteins may be differentially activated depending on the cell type or tissue. 2,[4][5][6] During myeloid differentiation, various isoforms of STAT3 and STAT5 are activated in a cell type-and maturation state-dependent manner. While STAT5 appears to be important for proliferative responses to interleukin-3 (IL-3), granulocyte-macrophage colonystimulating factor (GM-CSF), and G-CSF, 7,8 STAT3 is activated by G-CSF and appears to be the major STAT protein driving G-CSFmediated granulocytic differentiation. 9-16 Expression of dominant negative STAT3 9 or G-CSF receptor (G-CSFR) mutants lacking regions in the membrane distal domain, which contains recruitment sites for STAT3, [16][17][18][19][20] blocks granulocytic differentiation, demonstrating a requirement for STAT3 activation in growth arrest and morphologic granulocytic differentiation.Three distinct isoforms of STAT3, all derived from a single gene, have been identified 11,21 : STAT3␣ (p92), the full-length isoform expressed in most cells; STAT3␤ (p83), an alternatively spliced RNA form of STAT3␣ in which 55 amino acids of the C-terminal transactivation domain are replaced by 7 unique residues 22 ; and STAT3␥ (p72), also a C-terminal truncated form of STAT3␣ but derived posttranslationally th...

Activation of NF-κB and STAT3 in rat oval cells during 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration

“…In fact, a maturation state-dependent activation of STAT3 resulting in induction of specific cellular responses has been found during granulocytic differentiation. 50 Accordingly, we hypothesize that activation of STAT3 is necessary for the differentiation of oval cells along the hepatocyte lineage. However, because a link between the increased levels of phospho-STAT3 and the higher proliferative capacity of the cells also has been demonstrated, we cannot eliminate the possibility that STAT3 may drive proliferation of the expanding population of oval cells independently on their differentiation stage and may constitute a major force driving proliferation of the expanding population of oval cells.…”

The role of STAT3 in autophagy

“…17 Furthermore, a 72-kDa C-terminal-truncated form of STAT3 produced by the limited proteolysis of STAT3 during granulocytic differentiation also exists and is known as STAT3C/STAT3g, 18,19 and another 64-kDa truncated isoform known as STAT3d that is expressed in the early stage of granulocytic differentiation has been described. 20 The STAT3 protein shares similar functional domains with other STAT family members, including an amino-terminal coiled-coil domain beginning at residue 130, which enables protein-protein interactions, a central DNA-binding domain between residues 320 and 490, a linker domain extending from residues 490 to 580 that affects DNA-binding stability, a classic Src homology 2 (SH2) domain between residues 580 and 680, a tyrosine residue at 705 and a carboxyl transactivation domain, which has a serine phosphorylation site at Ser727 and is absent in the alternative splicing variant STAT3b 6,21 (Fig. 1).…”

IFN Regulatory Factor-2 Regulates Macrophage Apoptosis through a STAT1/3- and Caspase-1-Dependent Mechanism

“…Total RNA was isolated from liver samples, and 20 g of RNA was converted to cDNA with StrataScript reverse transcriptase (FairPlay Microarray Labeling Kit; Stratagene), using oligo(dT) [12][13][14][15][16][17][18] as a primer. cDNA was purified and labeled with Cy3 and Cy5 (Amersham Biosciences).…”

“…Among the STATs, STAT3 has been shown to be involved in apoptosis (11)(12)(13)(14)(15). Three distinct isoforms of STAT3 have been identified, as follows: STAT3␣ (p92), the full-length isoform expressed in most cells; STAT3␤ (p83), an alternatively spliced RNA form of STAT3␣ (16); and STAT3␥ (p72), a C-terminal truncated form of STAT3␣ derived posttranslationally through limited proteolysis (17). The ratio of the STAT3 isoforms appears to be related to the stage of cellular differentiation.…”

“…The ratio of the STAT3 isoforms appears to be related to the stage of cellular differentiation. STAT3␣ is more prevalent during early stages of granulocytic differentiation, whereas STAT3␤ is present later during maturation (17,18). Little is known about the function of STAT3␥.…”