Functional characterization of the L-type pyruvate kinase gene glucose response complex.

Abstract

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GIRE) of the L-PK gene is built around two noncanonical E boxes (element IA) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)-and COUPITF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GIRE function. Mutations transforming one or two noncanonical E boxes of element LA into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GIRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GIRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway. It is coordinately regulated at the transcriptional and posttranscriptional levels, positively by carbohydrates in the presence of insulin and negatively by glucagon via cyclic AMP (8,32). The regulatory region of the L-PK gene, responsible for its transcriptional response to carbohydrates and hormones, has been ascribed to a -120/ -183-bp proximal promoter fragment (2, 30), ex vivo by transient expression assays in hepatocytes in primary culture, and in vivo in transgenic mice (7). We had previously characterized the important cis-acting DNA elements in the L-PK gene promoter: in the 3'-5' direction upstream from the TATA box, we found, respectively, box Li, a binding site for hepatocyte nuclear factor 1 (HNF1); box L2, a binding site for nuclear factor 1; box L3, a binding site for HNF4; and box LA, a weak in vitro binding site for major late transcription factor (MLTF)/USF ( Fig. 1) (33). Box IA contains the L-PK gene glucose/insulin response element (GlRE) and is also indispensable for the action of glucagon and cyclic AMP. When L4 is oligomerized, it is able to confer glucose/insulin and cyclic AMP responsiveness on a

Hepatocyte Nuclear Factor-4 Is a Novel Downstream Target of Insulin via FKHR as a Signal-regulated Transcriptional Inhibitor

“…A number of studies have established that FKHR activates the transcription of gluconeogenic enzymes in the absence of insulin and inhibits the transcription in the presence of insulin. On the other hand, HNF-4 positively regulates the genes involved in glucose transport and glycolysis (4,6). Here, we provided a novel mechanism that insulin reversed the repression of HNF-4 transcriptional activity by FKHR.…”

Transcriptional control of genes that regulate glycolysis and gluconeogenesis in adult liver

“…The promoter (-183 to +11) contains binding sites for HNF-1, NF-I, HNF-4, upstream stimulating factor (USF)-related proteins and L5-binding factors (Figure 1) [80][81][82][83]. The HNF-4 site also binds chicken ovalbumin upstream promoter-transcription factor (COUP-TF)I and COUP-TFII [84]. The binding of HNF-4 apparently stabilizes NF-I binding [80].…”

Nuclear Factor 1 Family Members Interact with Hepatocyte Nuclear Factor 1α to Synergistically Activate L-type Pyruvate Kinase Gene Transcription

“…ChREBP is reported to bind to L-III as a heterodimer with Max-like protein X. It has been widely accepted that HNF4␣ is involved in transcriptional activation of the L-PK gene, because reporter gene assays and binding assays showed that HNF4␣ stimulates the L-PK promoter activity by binding to the L-IIB element (10,13,24). However, we suspected an alternative mechanism, because NF1 family members also bind to the L-IIB element as well as the accessory element in the S 14 gene, whereas HNF4␣ does not bind to this element in the S 14 gene (25).…”