Robin Wesselschmidt

Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells

We have generated H-2b mice with a homozygous null mutation in the granzyme (gzm) B gene. Gzm B is a neutral serine protease with Aspase activity that is found only in the granules of activated cytolytic T cells, natural killer cells, and lymphokine-activated killer cells. Gzm B-/- mice develop normally and have normal hematopoiesis and lymphopoiesis. In vitro, cytotoxic T lymphocytes (CTL) derived from gzm B-/- animals are able to induce 51Cr release from allotarget cells, but with reduced efficiency. However, gzm B-/- CTL have a profound defect in their ability to induce rapid DNA fragmentation and apoptosis in allogeneic target cells. This defect is kinetic since DNA fragmentation is partially compensated and 51Cr release is completely rescued with long incubation times. We conclude that gzm B serves a critical and nonredundant role for the rapid induction of target cell DNA fragmentation and apoptosis by alloreactive cytotoxic T lymphocytes.

Impaired Production and Increased Apoptosis of Neutrophils in Granulocyte Colony-Stimulating Factor Receptor–Deficient Mice

We have generated mice carrying a homozygous null mutation in the granulocyte colony-stimulating factor receptor (G-CSFR) gene. G-CSFR-deficient mice have decreased numbers of phenotypically normal circulating neutrophils. Hematopoietic progenitors are decreased in the bone marrow, and the expansion and terminal differentiation of these progenitors into granulocytes is impaired. Neutrophils isolated from G-CSFR-deficient mice have an increased susceptibility to apoptosis, suggesting that the G-CSFR may also regulate neutrophil survival. These data confirm a role for the G-CSFR as a major regulator of granulopoiesis in vivo and provide evidence that the G-CSFR may regulate granulopoiesis by several mechanisms. However, the data also suggest that G-CSFR-independent mechanisms of granulopoiesis must exist.

μ- and κ-Opioids Induce the Differentiation of Embryonic Stem Cells to Neural Progenitors

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected μ-opioid receptor (MOR-1) and κ-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [d-Ala2,MePhe4,Gly-ol5]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [d-Ala2,MePhe4,Gly-ol5]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.

Residual Cytotoxicity and Granzyme K Expression in Granzyme A-deficient Cytotoxic Lymphocytes

Cytotoxic lymphocytes contain granules that have the ability to induce apoptosis in susceptible target cells. The granule contents include perforin, a pore-forming molecule, and several granzymes, including A and B, which are the most abundant serine proteases in these granules. Granzyme B-deficient cytotoxic T lymphocytes (CTL) have a severe defect in their ability to rapidly induce apoptosis in their targets, but have an intact late cytotoxicity pathway that is in part perforin-dependent. In this report, we have created mice that are deficient for granzyme A and characterized their phenotype. These mice have normal growth and development and normal lymphocyte development, activation, and proliferation. Granzyme A-deficient CTL have a small but reproducible defect in their ability to induce 51Cr and125I-UdR release from susceptible allogeneic target cells. Since other granzyme A-like tryptases could potentially account for the residual cytotoxicity in granzyme A-deficient CTL, we cloned the murine granzyme K gene, which is linked to granzyme A in humans, and proved that it is also tightly linked with murine granzyme A. The murine granzyme K gene (which encodes a tryptase similar to granzyme A) is expressed at much lower levels than granzyme A in CTL and LAK cells, but its expression is unaltered in granzyme A−/− mice. The minimal cytotoxic defect in granzyme A−/− CTL could be due to the existence of an intact, functional early killing pathway (granzyme B dependent), or to the persistent expression of additional granzyme tryptases like granzyme K.

Mu and kappa opioids modulate mouse embryonic stem cell-derived neural progenitor differentiation via MAP kinases

J. Neurochem. (2010) 112, 1431–1441.

Cdc37 Regulates Ryk Signaling by Stabilizing the Cleaved Ryk Intracellular Domain

Ryk is a Wnt receptor that plays an important role in neurogenesis, neurite outgrowth, and axon guidance. We have reported that the Ryk receptor is cleaved by γ-secretase and that its intracellular domain (ICD) translocates to the nucleus upon Wnt stimulation. Cleavage of Ryk and its ICD is important for the function of Ryk in neurogenesis. However, the question of how the Ryk ICD is stabilized and translocated into the nucleus remains unanswered. Here, we show that the Ryk ICD undergoes ubiquitination and proteasomal degradation. We have identified Cdc37, a subunit of the molecular chaperone Hsp90 complex, as a Ryk ICD-interacting protein that inhibits proteasomal degradation of the Ryk ICD. Overexpression of Cdc37 increases Ryk ICD levels and promotes its nuclear localization, whereas Cdc37 knockdown reduces Ryk ICD stability. Furthermore, we have discovered that the Cdc37-Ryk ICD complex is disrupted during neural differentiation of embryonic stem cells, resulting in Ryk ICD degradation. These results suggest that Cdc37 plays an essential role in regulating Ryk ICD stability and therefore in Ryk-mediated signal transduction.