Lawrence D. Kerr

Inhibition of NF-kappaB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis.

In Drosophila, the Dorsal protein establishes the embryonic dorso–ventral axis during development. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-κB), is vital for the formation of the proximo–distal organizer of the developing limb bud, the apical ectodermal ridge (AER). Transcription of the NF-κB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-κB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-κB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-κB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

Reovirus-Induced Apoptosis Requires Activation of Transcription Factor NF-κB

ABSTRACT Reovirus infection induces apoptosis in cultured cells and in vivo. To identify host cell factors that mediate this response, we investigated whether reovirus infection alters the activation state of the transcription factor nuclear factor kappa B (NF-κB). As determined in electrophoretic mobility shift assays, reovirus infection of HeLa cells leads to nuclear translocation of NF-κB complexes containing Rel family members p50 and p65. Reovirus-induced activation of NF-κB DNA-binding activity correlated with the onset of NF-κB-directed transcription in reporter gene assays. Three independent lines of evidence indicate that this functional form of NF-κB is required for reovirus-induced apoptosis. First, treatment of reovirus-infected HeLa cells with a proteasome inhibitor prevents NF-κB activation following infection and substantially diminishes reovirus-induced apoptosis. Second, transient expression of a dominant-negative form of IκB that constitutively represses NF-κB activation significantly reduces levels of apoptosis triggered by reovirus infection. Third, mutant cell lines deficient for either the p50 or p65 subunits of NF-κB are resistant to reovirus-induced apoptosis compared with cells expressing an intact NF-κB signaling pathway. These findings indicate that NF-κB plays a significant role in the mechanism by which reovirus induces apoptosis in susceptible host cells.

Dynamic expression and activity of NF-κB during post-natal mammary gland morphogenesis

The Rel/NF-kappaB family of transcription factors has been implicated in such diverse cellular processes as proliferation, differentiation, and apoptosis. As each of these processes occurs during post-natal mammary gland morphogenesis, the expression and activity of NF-kappaB factors in the murine mammary gland were examined. Immunohistochemical and immunoblot analyses revealed expression of the p105/p50 and RelA subunits of NF-kappaB, as well as the major inhibitor, IkappaBalpha, in the mammary epithelium during pregnancy, lactation, and involution. Electrophoretic mobility shift assay (EMSA) demonstrated that DNA-binding complexes containing p50 and RelA were abundant during pregnancy and involution, but not during lactation. Activity of an NF-kappaB-dependent luciferase reporter in transgenic mice was highest during pregnancy, decreased to near undetectable levels during lactation, and was elevated during involution. This highly regulated pattern of activity was consistent with the modulated expression of p105/p50, RelA, and IkappaBalpha.

RAG2-/-, I kappa B-alpha-/- chimeras display a psoriasiform skin disease.

Nuclear factor-κB, a ubiquitous transcription factor involved in inflammatory and immune responses, is inappropriately activated in several immuno-related diseases, such as allograft rejection, or bronchial asthma. As nuclear factor-κB activity is regulated by inhibitor of κB (IκB), the gene encoding IκB-α was disrupted in mice to observe the in vivo effects of hyperactivation of nuclear factor-κB. IκB-α–/– mice have constitutive nuclear factor-κB activity, severe skin disease, and neonatal lethality. To determine the role of IκB-α deficient immunocytes in the pathogenesis of the skin disease in adult mice, we utilized the RAG2-deficient blastocyst complementation system to generate RAG2–/–, IκB-α–/– chimeras. These animals display a psoriasiform dermatitis characterized by hyperplastic epidermal keratinocytes and dermal infiltration of immunocytes, including lymphocytes. Skin grafts transferred from diseased chimeras to recipient nude mice produce hyperproliferative psoriasiform epidermal keratinocytes in response to stimulation. Furthermore, adoptive transfer of lymph node cells from diseased chimeras to RAG2–/– recipient mice recapitulates the disease. Taken together, these characterizations provide evidence to suggest that constitutive activation of nuclear factor-κB, due to deficiency in IκB-α, can invoke severe psoriasiform dermatitis in adult mice.

RAG2–/–, IκB-α–/– Chimeras Display a Psoriasiform Skin Disease

Nuclear factor-κB, a ubiquitous transcription factor involved in inflammatory and immune responses, is inappropriately activated in several immuno-related diseases, such as allograft rejection, or bronchial asthma. As nuclear factor-κB activity is regulated by inhibitor of κB (IκB), the gene encoding IκB-α was disrupted in mice to observe the in vivo effects of hyperactivation of nuclear factor-κB. IκB-α–/– mice have constitutive nuclear factor-κB activity, severe skin disease, and neonatal lethality. To determine the role of IκB-α deficient immunocytes in the pathogenesis of the skin disease in adult mice, we utilized the RAG2-deficient blastocyst complementation system to generate RAG2–/–, IκB-α–/– chimeras. These animals display a psoriasiform dermatitis characterized by hyperplastic epidermal keratinocytes and dermal infiltration of immunocytes, including lymphocytes. Skin grafts transferred from diseased chimeras to recipient nude mice produce hyperproliferative psoriasiform epidermal keratinocytes in response to stimulation. Furthermore, adoptive transfer of lymph node cells from diseased chimeras to RAG2–/– recipient mice recapitulates the disease. Taken together, these characterizations provide evidence to suggest that constitutive activation of nuclear factor-κB, due to deficiency in IκB-α, can invoke severe psoriasiform dermatitis in adult mice.

Protein kinase C isotypes required for phorbol-ester induction of stromelysin-1 in rat fibroblasts

The phorbol‐ester tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) is a potent inducer of the metalloproteinase stromelysin in fibroblasts in vivo and in several cultured cell lines. Rat‐1 and Rat‐2 fibroblasts, however, do not respond to TPA stimulation by induction of stromelysin gene activity, although collagenase promoter‐mediated activity is induced threefold by TPA treatment in these cells. We determined that rat fibroblasts expressed protein kinase C (PKC) α, PKCδ, PKCϵ, and PKCζ but neither the mRNA nor the protein for PKCβ. When Rat‐2 fibroblasts were stably transfected with an expression vector producing PKCβ, however, TPA treatment of these variants resulted in a 3.1‐fold induction of stromelysin promoter‐mediated luciferase activity compared with a 1.3‐fold induction in parental Rat‐2 cells (P < 0.002). Transient transfection of PKCϵ produced a small but significant increase in TPA‐stimulation of both stromelysin‐ and collagenase‐mediated gene expression. These results suggest that there are PKC isotype‐specific signaling pathways that can differentially regulate matrix metalloproteinase gene expression.

RAG2|[ndash]||[sol]||[ndash]|, I|[kappa]|B-|[alpha]||[ndash]||[sol]||[ndash]| Chimeras Display a Psoriasiform Skin Disease

Nuclear factor-κB, a ubiquitous transcription factor involved in inflammatory and immune responses, is inappropriately activated in several immuno-related diseases, such as allograft rejection, or bronchial asthma. As nuclear factor-κB activity is regulated by inhibitor of κB (IκB), the gene encoding IκB-α was disrupted in mice to observe the in vivo effects of hyperactivation of nuclear factor-κB. IκB-α–/– mice have constitutive nuclear factor-κB activity, severe skin disease, and neonatal lethality. To determine the role of IκB-α deficient immunocytes in the pathogenesis of the skin disease in adult mice, we utilized the RAG2-deficient blastocyst complementation system to generate RAG2–/–, IκB-α–/– chimeras. These animals display a psoriasiform dermatitis characterized by hyperplastic epidermal keratinocytes and dermal infiltration of immunocytes, including lymphocytes. Skin grafts transferred from diseased chimeras to recipient nude mice produce hyperproliferative psoriasiform epidermal keratinocytes in response to stimulation. Furthermore, adoptive transfer of lymph node cells from diseased chimeras to RAG2–/– recipient mice recapitulates the disease. Taken together, these characterizations provide evidence to suggest that constitutive activation of nuclear factor-κB, due to deficiency in IκB-α, can invoke severe psoriasiform dermatitis in adult mice.