Taken together, the data presented here demonstrate that GATA4 has a crucial role in controlling jejunal physiology. The first indicator of disrupted intestinal function in GATA4 mutant mice was the finding that such mice were consistently smaller than control littermates. Considering that lipid absorption was impaired in mice lacking GATA4 in the intestinal epithelium, the fact that the weight difference did not reach statistical significance at later time points may reflect the change in diet at weaning from a high-fat diet (milk) to a low-fat diet (standard rodent chow). Indeed, we compared control and mutant pups at 1 week and 2 weeks of age and found that mutant pups weighed less at both timepoints (1wk control 5.35±0.22g n=16; mutant 4.11±0.24g, n=6, (p<0.01); 2wk control 8.25±0.21g, n=6; mutant 6.66± 0.19, n = 5, (p < 0.01)). In further support of this difference resulting from a metabolic cause, we showed that mutant animals consumed the same amount as control animals ruling out the possibility that Gata4 conditional knockout animals were smaller simply because they consumed less food.
The reductions in circulating cholesterol and phospholipids and in dietary fat and cholesterol uptake in Gata4
conditional knockout animals compared with controls indicated that both lipid and cholesterol metabolism were severely disrupted in GATA4 mutant mice. Although we observed a small but statistically significant (p < 0.05) decrease in goblet cells, we do not believe that this would contribute to a defect in absorption since nutrient uptake is accomplished exclusively by enterocytes. In contrast, our observation of altered villous morphology in GATA4 null jejunum raises the possibility that reduced villous length resulted in decreased lipid and cholesterol absorption. However, protein tyrosine kinase 6
) null mice, which have jejunal villi that are twice the length of control villi, failed to absorb any more fat than wild-type animals.15
Expression of Ptk6
was increased 3.6 fold in GATA4 mutant jejunum compared with control jejunum providing a potential explanation for the shorter jejunal villi observed in the absence of GATA4 (Table 4, supplemental information
). Increased jejunal expression of Ptk6
, which is more abundant in the ileum compared with the jejunum,15
further supports our conclusion that the identity of GATA4 null jejunum shifted toward that of the ileum.
The exact mechanism through which loss of GATA4 so dramatically affects lipid and cholesterol uptake likely involves both direct and indirect mechanisms. Based on our gene array data, however, we believe it is reasonable to propose that GATA4 plays an essential role in the regulation of a plethora of genes whose combined action is required for normal jejunal cholesterol and fat absorption. Decreased expression of genes encoding fat and cholesterol receptors and transporters such as Slc27a2
, and Scarb1
provide one molecular mechanism to explain the observed phenotype. The Slc27a2
gene encodes a fatty acid transporter that caused increased fat uptake when over-expressed in cell lines.16
Although defining the role that the receptors CD36 and SCARB1 play in intestinal lipid and cholesterol uptake has been somewhat controversial because Cd36
knockout mice failed to show a conclusive defect in intestinal fatty acid and cholesterol uptake,17, 18
new studies support the conclusion that both receptors directly participate in these processes. Specifically, transgenic mice over-expressing Scarb1
showed accelerated lipid and cholesterol absorption by the intestine,19
and purified brush border membrane vesicles incubated with an antibody against SCARB1 failed to bind cholesterol with high affinity.20
Moreover, both Nassir et al.21
and Drover et al
recently demonstrated that CD36 serves as a receptor for dietary fat and cholesterol absorption in the proximal intestine.
Bile acid homeostasis also plays a key role in lipid and cholesterol metabolism.23
Bile acids function in the jejunum to emulsify lipids and cholesterol thereby enabling absorption of these nutrients. Most intestinal bile acid absorption occurs in the ileum, a site normally lacking GATA4. We found that loss of GATA4 in the jejunum, however, creates a molecular environment in which genes encoding multiple critical components of the bile acid absorption pathway are improperly induced. We detected high-level expression of Slc10a2
, which encodes the primary ileal bile acid transporter (ASBT), in GATA4 null jejunum suggesting that bile acids were prematurely absorbed in the jejunum of mutant mice. Uptake of bile acids results in activation of an FXR-driven pathway that culminates in repression of bile acid biosynthesis by the liver.24, 25
Although Nr1h4 (Fxr
) expression was up-regulated only 2 fold in GATA4 null jejuna compared with controls, its activity was strongly induced as was demonstrated by increased expression of FXR target genes including Fabp6
, Fgf15, Osta,
. In fact, Fabp6
were undetectable in control jejunum. Moreover, down-regulation of Cyp7a1
expression in livers of GATA4 intestinal-specific knockout mice compared with controls provides further evidence that loss of GATA4 in the jejunum disrupted enterohepatic signaling.
In summary, elimination of GATA4 from the intestinal epithelium severely disrupted jejunal physiology. We propose that both decreases in expression of key fat and cholesterol transporters and the activation of bile acid absorption in jejunal enterocytes lacking GATA4 cause the dramatic decrease in lipid and cholesterol absorption observed. In future experiments, it will be important to determine the extent to which changes in receptor and transporter gene expression and changes in bile acid homeostasis each contribute to the observed defect in lipid metabolism. Our data agree with and expand upon the work of Bosse et al.2
who suggested that GATA4 is required to maintain jejunal-ileal identities based on changes in expression of a limited set of jejunal and ileal specific genes. The data we present definitively demonstrate that the absence of GATA4 in the jejunum results in a loss of jejunal function and a concomitant gain in ileal function. In addition to physiological changes indicative of a loss of the jejunal domain and an extension of the ileal domain, comparison of the global gene expression profiles among control jejunum, control ileum, and GATA4 null jejunum demonstrated a wide-scale shift in gene expression in mutant mice from that specifically associated with jejunum to that of ileum. Future studies in which GATA4 is ectopically expressed in the ileum should provide insight into the extent to which GATA4 plays a dominant role in conferring jejunal character upon the intestine.