Stephen Koch

Human hexokinase II mRNA and gene structure.

This study reports the isolation and characterization of the human hexokinase II (HKII) gene. This gene is ∼ 50 kilobases in length and contains 18 exons, ranging in size from 96 to 2,536 base pairs, that are exactly the same size as the corresponding exons in the rat HKII gene. A cDNA representing the entire open reading frame for HKII was synthesized using a series of polymerase chain reactions with human skeletal muscle RNA as the template, and this allowed us to deduce the complete structure of the HKII mRNA. The human HKII mRNA has 431 nucleotides (nt) of 5′ noncoding sequence, 2,751 nt of coding sequence, and 2,394 nt of 3′ noncoding sequence. The open reading frame encodes a protein of 917 amino acids with an estimated molecular mass of 102.4 kDa. There is a high degree of similarity in the amino acid and nt sequences of the rat and human glucokinase and HKII proteins and genes. This, coupled with the observation that the exon sizes are conserved, suggests a common evolutionary origin of the these two genes.

Insulin Decreases H4IIE Cell PEPCK mRNA by a Mechanism That Does Not Involve cAMP

Insulin is thought to influence some metabolic events by decreasing the intracellular concentration of cyclic AMP (cAMP). To test whether this explains the repression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) by insulin we measured intracellular cAMP, cAMP-dependent protein kinase; mRNAPEPCK, and PEPCK gene transcription in cultured Reuber H4IIE hepatoma cells treated with forskolin with and without insulin. In untreated cells, the concentration of cAMP was 2.9 pmol/mg of protein. Forskolin at 1,10, and 50 μM increased the level of cAMP to 9.2, 35.8, and 115 pmol/mg of protein, respectively; 5 nM insulin had no significant effect on these cAMP concentrations. In untreated cells, the activity ratio of cAMP-dependent protein kinase was 0.43, and 50 μM forskolin increased this to 0.96; insulin had no effect on this ratio at times from 15–180 min. In untreated cells mRNAPEPCK bound 15 cpm of a 32P-labeled cDNA probe per microgram of total cellular RNA. Forskolin, at 1, 10, and 50 μM, increased this to 48, 96, and 115 cpm/μg RNA. Insulin (5 nM), in combination with 0, 1, 10, and 50 μM forskolin, decreased the concentration of mRNAPEPCK to 5, 8, 23, and 29 cpm/μg RNA, respectively. Finally, the rate of transcription of the PEPCK gene was 85, 168, 630, 823, and 884 parts per million (ppm) in H4IIE cells treated for 30 min with 0, 1, 5, 10, and 50 μM forskolin, respectively, while the corresponding rates in the presence of 5 nM insulin were 49, 45, 84, 85, and 136 ppm. These results demonstrate that insulin represses mRNAPEPCK and PEPCK gene transcription in H4IIE cells by a mechanism that is independent of the regulation of intracellular cAMP concentration and cAMP-dependent protein kinase activity.

26S proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells.

Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

Monophosphoryl lipid A inhibits the cytokine response of endothelial cells challenged with LPS

Monophosphoryl lipid A (MPLA) is a TLR4 agonist that is used as an immunomodulator in human vaccines; additionally, it has been shown to be protective in models of sepsis. As endothelial cells regulate inflammation, we hypothesized that MPLA would decrease activation of human umbilical vein endothelial cells (HUVECs) to LPS. We studied HUVECs challenged with LPS (100 ng/ml), MPLA (0.001–100 µg/ml) or a combination. Secretion of IL-6, RANTES (CCL5) and IP-10 (CXCL10) were assessed by ELISA. Activation of MAPK phosphorylation and cytokine transcription were assessed by Western blot analysis and PCR, respectively. MPLA alone was a weak stimulator of myeloid differentiation primary response protein 88-dependent IL-6 and did not induce TIR-domain-containing adapter-inducing IFN-β (TRIF)-dependent chemokine responses. MPLA significantly reduced LPS-mediated IL-6 production. This inhibitory effect was also conferred for the TRIF-dependent chemokines RANTES and IP-10. Inhibition of LPS-mediated activation by MPLA was associated with reduced p38 phosphorylation and mRNAs encoding inflammatory cytokines. MPLA inhibition of LPS signaling appeared to be at the level of the TLR4 receptor, acting as a receptor antagonist with weak agonistic properties. This study provides evidence of a novel mechanism for the inhibitory effect of MPLA on LPS-induced endothelial activation.

Isolation, characterization and chromosomal localization of a human pseudogene for hexokinase II.

A processed pseudogene for hexokinase II (HKII), the first such reported for a member of the hexokinase gene family, was isolated from a human genomic library by using a rat HKII cDNA as a probe. The pseudogene contains a region that is identical to the open reading frame of the human HKII cDNA at 97% of the nucleotide positions, but it contains several frameshift mutations, small deletions and insertions, and several stop codons. The human HKII pseudogene is located on the X chromosome and is integrated into a long interspersed nuclear repetitive DNA element (LINE). We estimate that this integration event occurred approximately 14-16 Myr (million years) ago.

Potentiation and tolerance of toll-like receptor priming in human endothelial cells

&NA; Repeated challenge of lipopolysaccharide (LPS) alters the response to subsequent LPS exposures via modulation of toll‐like receptor 4 (TLR4). Whether activation of other TLRs can modulate TLR4 responses, and vice versa, remains unclear. Specifically with regards to endothelial cells, a key component of innate immunity, the impact of TLR cross‐modulation is unknown. We postulated that TLR2 priming (via Pam3Csk4) would inhibit TLR4‐mediated responses while TLR3 priming (via Poly I:C) would enhance subsequent TLR4‐inflammatory signaling. We studied human umbilical vein endothelial cells (HUVECs) and neonatal human dermal microvascular endothelial cells (HMVECs). Cells were primed with a combination of Poly I:C (10 &mgr;g/ml), Pam3Csk4 (10 &mgr;g/ml), or LPS (100 ng/ml), then washed and allowed to rest. They were then rechallenged with either Poly I:C, Pam3Csk4 or LPS. Endothelial cells showed significant tolerance to repeated LPS challenge. Priming with Pam3Csk4 also reduced the response to secondary LPS challenge in both cell types, despite a reduced proinflammatory response to Pam3Csk4 in HMVECs compared to HUVECs. Poly I:C priming enhanced inflammatory and interferon producing signals upon Poly I:C or LPS rechallenge, respectively. Poly I:C priming induced interferon regulatory factor 7, leading to enhancement of interferon production. Finally, both Poly I:C and LPS priming induced significant changes in receptor‐interacting serine/threonine‐protein kinase 1 activity. Pharmacological inhibition of receptor‐interacting serine/threonine‐protein kinase 1 or interferon regulatory factor 7 reduced the potentiated phenotype of TLR3 priming on TLR4 rechallenge. These results demonstrate that in human endothelial cells, prior activation of TLRs can have a significant impact on subsequent exposures and may contribute to the severity of the host response.