β1,4-Galactosyltransferase 1 Is a Key Regulator of Thrombopoiesis

Sialylated derivatives of N-Acetyl Lactosamine (LacNAc) regulate platelet lifespan, hepatic thrombopoietin (TPO) production, and thus thrombopoiesis. A major enzyme that synthesizes LacNAc is β1,4-galactosyltransferase 1 (β4GalT1), the gene of which is highly conserved through evolution. Long considered a housekeeping gene, the transcriptional regulation of B 4GALT1 remained widely understudied, and recent observations have shown that itspromoter region is rich in enhancer sequences.

Both TPO and the chemokine CXCL12 regulate thrombopoiesis. Here we report that TPO and CXCL12 regulate megakaryocyte (MK) function by increasing β 4galt1 transcription . TPO and CXCL12 induced a two-wave expression of LacNAc in MKs. TPO was required for LacNAc production in wild type (WT) MKs, and β4GalT1 deletion in mice (β4GalT1^-/-) resulted in severe thrombocytopenia due to increased integrin activity in MKs, and defective MK localization within the bone marrow (BM).

Detailed analysis of β4GalT1^-/- MKs and platelets showed that β4GalT1^-/- MKs are almost completely devoid of LacNAc expression. Complete lack of galactosyltransferase activity in β4GalT1^-/- MKs and platelets was demonstrated using in vitro enzymatic assays, showing that LacNAc synthesis in MKs and platelets depends primarily on β4GalT1. In WT MKs cultured in the presence of TPO alone, and in isolated circulating platelets, LacNAc expression was particularly evident in polypeptides with molecular weight ranging between 100-150 kDa, which were identified as αIIbβ3, GPIbα and β1 integrin. Addition of CXCL12 in vitro increased LacNAc expression specifically on a 130-kDa polypeptide, identified as the β1 integrin subunit. Further detailed investigation of receptor expression and function showed that WT MKs expressed two β1 integrin glycoforms of 130 and 100 kDa, and WT platelets expressed only the 130-kDa glycoform. β4GalT1^-/- MKs and platelets expressed only the immature 100-kDa β1 integrin glycoform. While the subcellular distribution β1 integrin was indistinguishable between WT and β4GalT1^-/- MKs and platelets, lack of LacNAc synthesis resulted in increased of β1 integrin surface expression, as determined by flow cytometry. β4GalT1^-/- platelet adhesion to collagen and laminin was significantly increased and was unaffected by addition of RGDS. By contrast, GPIbα and αIIbβ3 expression and function were not affected by β4GalT1 loss.

Together, the results suggest that TPO and CXCL12 upregulate β4GalT1-dependent LacNAc synthesis to promote thrombopoiesis, likely regulating MK β1 integrin expression, glycosylation and subsequent interaction with components of the extracellular matrix. Investigating the mechanisms regulating posttranslational modifications induced by various hematopoietic stimulating chemokines and cytokines will contribute to better understand thrombopoiesis.