Platelet Protein Kinase C-θ Deficiency With Human RUNX1 Mutation

Abstract

Objective Mutations in hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1 we have described decreased platelet pleckstrin phosphorylation and protein kinase C-θ (PKC-θ, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-θ in megakaryocytes/platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. Methods and Results In chromatin immunoprecipitation assay using megakaryocytic cells there was RUNX1 binding in vivo to PRKCQ promoter region −1225/−1056 bp containing a RUNX1 consensus site ACCGCA at −1088/−1069 bp; electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-θ protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC-θ protein were decreased by siRNA knockdown of RUNX1. Conclusion Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC-θ deficiency associated with RUNX1 haplodeficiency.

Defective acid hydrolase secretion in RUNX1 haplodeficiency: Evidence for a global platelet secretory defect

“…Moreover, our expression profiling studies in the same patient showed that a wide array of genes involved in platelet function and formation are downregulated [8]. We have shown that several relevant genes including PRKQ [9] , MYL9 [10], ALOX12 [12], PLDN [24] and PCTP [25] are direct transcriptional targets of RUNX1. Thus, we postulate that the secretory abnormalities result from aberrations in common pathways critical to the general process of exocytosis, for example, involving vesicle transport mechanisms, Rab proteins, and SNARE proteins [16, 26].…”

“…In addition, abnormalities in mechanisms that regulate secretion have been reported in some patients [2, 3, 5], including in myosin light chain and pleckstrin phosphorylation, and decreased platelet PKC-θ [6, 7]. Platelet mRNA expression profiling of a patient with RUNX1 mutation [8] and other studies [7] have shown downregulation of several genes, including MYL9 , PRKQ ALOX12 and PF4, and some of these are direct transcriptional targets of RUNX1, providing evidence for aberrations in multiple platelets mechanisms, including in those regulating platelet responses to activation [2, 9–12]. …”

Inherited platelet dysfunction and hematopoietic transcription factor mutations

“…In our platelet transcript profiling studies of a patient with RUNX1 mutation numerous genes relevant to multiple platelet biological pathways were downregulated. 33 Several genes have been shown to be direct transcriptional targets of RUNX1, including 12-lipoxygenase ( ALOX12 ), 25 platelet factor 4 ( PF4 ), 34 platelet myosin light chain ( MYL9 ), 35 protein kinase C-θ ( PRKCQ ), 36 palladin ( PLDN ), 26 and the thrombopoietin receptor ( c-MPL ). 37 These impact various aspects of MK biology, platelet production, structure, signaling, and responses.…”

Mechanisms underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: potential role for candidate RUNX1 targets

“…RUNX1 targets involved in decreased platelet production include the genes for the MPL receptor (MPL) [11], myosin IIA (MYH9), myosin IIB (MYH10), and myosin regulatory light chain (MYL9) [10]. Besides MYL9, other RUNX1 target genes with a role in platelet function have been found, by platelet expression profiling, to be downregulated in one FPD/AML patient; these included the genes for platelet 12-lipoxygenase (ALOX12) and protein kinase C-h (PKCh) [12][13][14][15][16].…”