Abstract
Activin receptor-like kinase (ALK)7 is a type I serine/threonine kinase receptor of the transforming growth factor (TGF)-beta family of proteins that has similar properties to other type I receptors when activated. To see whether ALK7 can induce apoptosis as can some of the other ALK proteins, we infected the FaO rat hepatoma cell line with adenovirus expressing a constitutively active form of the ALK7. Cells infected with active ALK7 adenovirus showed an apoptotic-positive phenotype, as opposed to those that were infected with a control protein. DNA fragmentation assays and fluorescence-activated cell sorter analysis also indicated that ALK7 infection induced apoptosis in FaO cells. We also confirmed this finding in Hep3B human hepatoma cells by transiently transfecting the constitutively active form of ALK7, ALK7(T194D). Investigation into the downstream targets and mechanisms involved in ALK7-induced apoptosis revealed that the TGF-beta signaling intermediates, Smad2 and -3, were activated, as well as the MAPKs JNK and p38. In addition, caspase-3 and -9 were also activated, and cytochrome c release from the mitochondria was observed. Short interfering RNA-mediated inhibition of Smad3 markedly suppressed ALK7-induced caspase-3 activation. Treatment with protein synthesis inhibitors or the expression of the dominant-negative form of the stress-activated protein/extracellular signal-regulated kinase 1 abolished not only JNK activation but apoptosis as well. Taken together, these results suggest that ALK7 induces apoptosis through activation of the traditional TGF-beta pathway components, thus resulting in new gene transcription and JNK and p38 activation that initiates cross-talk with the cellular stress death pathway and ultimately leads to apoptosis.
Highlights
The TGF-β superfamily of cytokines is responsible for regulating a wide range of cellular responses in many different cell types, including differentiation, cell growth, and apoptosis [1,2,3,4,5] TGF-βs signal through a serine/theorine kinase pathway that begins upon ligand binding to a set of two transmembrane receptors, termed type I or type II, located on the surface of the cell plasma membrane [1]
Constitutive active ALK7 in Fao Cells results in Apoptosis In order to investigate the possible role ALK7 plays in apoptosis, a constitutive active mutant form bearing a HA-tag was inserted into an adenovirus expression system and used for infection into cultures of FaO cells
As a final test to see if ALK7 induces apoptosis in FaO cells, a FACS analysis was conducted of cells that had been infected with LacZ, or the active forms of either ALK5 or ALK7
Summary
The TGF-β superfamily of cytokines is responsible for regulating a wide range of cellular responses in many different cell types, including differentiation, cell growth, and apoptosis [1,2,3,4,5] TGF-βs signal through a serine/theorine kinase pathway that begins upon ligand binding to a set of two transmembrane receptors, termed type I or type II, located on the surface of the cell plasma membrane [1]. The type II receptor is responsible for initial ligand binding, which acts to recruit and activate, via phosphorylation, the type I receptor. The type I receptor phosphorylates a set of proteins named Smads that are specific for each kind of type I receptor. The function of ALK7 as a type I receptor was confirmed with a constitutively active mutant form that activated a TGF-β/Activin response reporter [6]. In the rat pheochromocytoma PC12 cell line, ALK7 activated both samd 2 and smad 3, but the map kinases of ERK and JNK, as well as inhibiting cell proliferation [9]. The human gene for ALK7 has been mapped to the genetic location of 2q24.1-q3, with most of the mRNA located in the brain, pancreas, and colon. The human gene for ALK7 has been mapped to the genetic location of 2q24.1-q3, with most of the mRNA located in the brain, pancreas, and colon. [10] Recently the human form of ALK7 has been identified along with three splice variants that are expressed in the placenta throughout various stages of pregnancy [11]
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