Activin βC and βE Genes Are Not Essential for Mouse Liver Growth, Differentiation, and Regeneration

Growth factors and hormones play an extremely important role in regulating biological processes from patterning of the early embryo to regulating the function of tissues and organs. The largest family of growth factors is the transforming growth factor β (TGF-β) superfamily of secreted dimeric proteins (12). Members of this family include activins, TGF-βs, bone morphogenetic proteins (BMPs), and growth differentiation factors and demonstrate diverse functions including roles in left-right asymmetry, skeletal development, reproduction, and oncogenesis (29, 52). Both activins (β-β dimers) and inhibins (α-β dimers) have historically been shown to be regulators of follicle-stimulating hormone (FSH) secretion from the pituitary gland (54). These earlier results were later confirmed by in vivo analysis of activin βB-, activin receptor type IIA (ActRIIA)-, and α-inhibin-deficient mice (32–34). In studies involving Xenopus laevis oocytes (36), activins were tested for their ability to induce mesoderm formation. However, this observation is not true for mice (35), suggesting that the results obtained with X. laevis oocyte injection experiments may be due to nonphysiological effects of the activin ligands. The adult liver detoxifies the blood through the actions of various enzymes, synthesizes normal serum proteins such as the acute-phase proteins and albumin, and produces bile, which is critical for normal fat absorption (9). Activin A and TGF-β1 have been shown to affect liver growth and function. These two proteins can inhibit mitogen-induced DNA synthesis in hepatocytes (5, 41, 51, 57), induce hepatocellular apoptosis in vitro (7, 21, 42, 48), and stimulate glycogenolysis from cultured hepatocytes (39). In vivo, pharmacological levels (e.g., intravenous infusion of recombinant activin A [21, 48]) or pathophysiologically high levels of activins (8, 30, 32) cause a reduction in liver mass by inducing hepatocellular necrosis around the central vein. Liver regeneration occurs following liver injury that results in loss of liver mass. The liver regenerates by a process of hypertrophy and a near-synchronous proliferation of the remaining cells through several cycles of replication. Several cytokines are thought to play early roles in the regeneration process: interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (reviewed in reference 38). IL-6 has been shown to play a critical role in the progression of liver regeneration but not for initiation (10). While the interleukins and tumor necrosis factor alpha are thought to be important during the early stages of liver regeneration, TGF-β1 to -3 and activins are thought to be negative regulators during this process (27, 38), since mRNAs encoding these ligands are up-regulated during this process (3, 57). However, this latter hypothesis has not yet been tested in vivo. Recently, our groups (15, 28) and others (19, 43, 47) have cloned three new members of the TGF-β superfamily which demonstrate highest amino acid identity in the mature peptide region to the activin (βA and βB) subfamily. Two of these new members, designated activin βC (actβC) and activin βE (actβE), have been cloned in mammals (15, 19, 28, 47), while the third, activin βD, has been found only in X. laevis (43). Weak induction of a secondary axis has been observed when activin βD mRNA is injected into the ventral blastomeres of Xenopus embryos (43), but activin βC and βE have not yet been functionally tested by any in vitro or in vivo bioassays. Expression of actβC and actβE is primarily liver specific in the adult (14, 28, 47), unlike activin βA and activin βB, which are widely expressed in multiple tissues in rodents (16, 37) and humans (53). Based on the highly restricted tissue expression pattern, we hypothesized that activins βC and βE may play critical roles in liver physiology. To compare the in vivo functions of these novel liver-restricted mammalian activin βC and βE genes to those of the known activin βA and βB genes, we generated null mutations in actβC, actβE, or both genes in mice. Our studies show that activin βC and βE are not essential for liver development, liver function, or reproduction.