Jianxun Lei

Identification and characterization of a new splicing variant of vascular endothelial growth factor: VEGF183

We report the discovery of a new splicing variant of vascular endothelial growth factor named VEGF183. It is six amino acids shorter than its closest relative, VEGF189, due to the utilization of a conserved alternate splicing donor site within exon 6a. Highly expressed in heart tissue, VEGF183 is detected in transiently transfected COS cells as 28-32-kDa monomers under reduced condition, and 46-kDa dimers under non-reduced condition - the functional unit for all VEGF isoforms.

Expression, purification and charaterization of recombinant mouse MT5‐MMP protein products

We have recently identified the fifth member of the membrane‐type matrix metalloproteinase subfamily, MT5‐MMP/MMP24, which is expressed in a brain specific manner (Duanqing Pei (1999) J. Biol. Chem. 274, 8925–8932). To further characterize its enzymic properties, an expression construct was engineered to produce MT5‐MMP as a soluble and active form by truncating its transmembrane domain. Stable expression cell lines were subsequently established from MDCK cells transfected with this construct. Unfortunately, purification of MT5‐MMP from the culture media in large quantity proves to be difficult initially due to its rapid turnover via a mechanism which can be inhibited by a broad spectrum metalloproteinase inhibitor, BB94. Thus, BB94 was included in the cell culture medium and throughout the purification process except the final step of chromatography to protect MT5‐MMP from destruction. Purified to homogeneity and free of the synthetic inhibitor, MT5‐MMP can activate progelatinase A efficiently in a TIMP2 sensitive fashion. A preliminary screen for its potential substrates among extracellular matrix components identified the proteoglycans as the preferred substrates for MT5‐MMP. Furthermore, it is determined that the stability of purified MT5‐MMP is temperature dependent with rapid destruction at 37°C, but being relatively stable at temperatures 4°C or lower. These observations establish MT5‐MMP as a proteoglycanase with a short half‐life at body temperature, which may be critical for tightly controlled turnover of ECM components such as those in the brain.

T3 increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent pathway in rat adult alveolar epithelial cells

Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and beta-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase alpha(1)-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.

Thyroid hormone rapidly stimulates alveolar Na,K-ATPase by activation of phosphatidylinositol 3-kinase.

Purpose of reviewNongenomic actions of 3,3′,5-triiodo-L-thyronine (T3) occur quite rapidly usually via activation of signaling cascades. In this review, we focus on recent advances made in the understanding of activation of the phosphatidylinositol 3-kinase pathway by T3 in alveolar epithelial cells, resulting in upregulation of Na,K-ATPase hydrolytic activity and potential physiological significance of this finding. Recent findingsT3 stimulates the Src family of kinases. Activation of Src-kinase and phosphatidylinositol 3-kinase/protein kinase B is required for the T3-induced stimulation of alveolar epithelial Na,K-ATPase activity in rat alveolar epithelial cells. The stimulation does not require transcription. This T3-sensitive Na,K-ATPase stimulation in rat alveolar epithelial cells is switched on late in gestation. In skin fibroblasts phosphatidylinositol 3-kinase is also involved in the nongenomic T3 stimulation of ZAK1-4α protein expression, an endogenous calcineurin inhibitor. SummaryT3 plays an important role in cell survival and differentiation. Nongenomic regulation of phosphatidylinositol 3-kinase and downstream molecules by T3 is being recognized in different tissues. Upregulation of alveolar Na,K-ATPase is one such molecule, which plays an important role in removal of edema fluid from the alveolar space. These effects are rapid and do not require direct nuclear gene transcription.

Cannabinoid receptor-specific mechanisms to alleviate pain in sickle cell anemia via inhibition of mast cell activation and neurogenic inflammation.

Sickle cell anemia is a manifestation of a single point mutation in hemoglobin, but inflammation and pain are the insignia of this disease which can start in infancy and continue throughout life. Earlier studies showed that mast cell activation contributes to neurogenic inflammation and pain in sickle mice. Morphine is the common analgesic treatment but also remains a major challenge due to its side effects and ability to activate mast cells. We, therefore, examined cannabinoid receptor-specific mechanisms to mitigate mast cell activation, neurogenic inflammation and hyperalgesia, using HbSS-BERK sickle and cannabinoid receptor-2-deleted sickle mice. We show that cannabinoids mitigate mast cell activation, inflammation and neurogenic inflammation in sickle mice via both cannabinoid receptors 1 and 2. Thus, cannabinoids influence systemic and neural mechanisms, ameliorating the disease pathobiology and hyperalgesia in sickle mice. This study provides ‘proof of principle’ for the potential of cannabinoid/cannabinoid receptor-based therapeutics to treat several manifestations of sickle cell anemia.

Nongenomic actions of l-thyroxine and 3,5,3′-triiodo-l-thyronine. Focus on “l-Thyroxine vs. 3,5,3′-triiodo-l-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase”

the molecular mechanisms of the numerous cellular actions of thyroid hormone have been widely studied. The classical mechanism of thyroid hormone action occurs by uptake of l-thyroxine (T4) or 3,5,3′ triiodo-l-thyronine (T3) into cells, transport into the cell nucleus, binding with a thyroid receptor (TR), recruitment of coactivators, and regulation of gene transcription via thyroid response elements (TRE). T3 is more potent in these actions than T4. These genomic actions require access of the hormone to the cell interior, translocation to the nucleus, alteration of the rate of gene transcription, and translation of the specific gene product; thus, the overall response generally requires several hours to become manifest. Over the past decade, many actions of thyroid hormone have been described that do not involve initial nuclear action of thyroid receptors and/or gene transcription; therefore they are considered “nongenomic” (6). Davis and colleagues (6, 7) have described both TR-dependent and TR-independent novel nongenomic actions that involve cell surface receptors and signal transduction pathways. Some actions that begin nongenomically at the cell surface may ultimately become nuclear and cellular events. One example is the phosphorylation of the TRβ by T4 that results in derepression of the transcriptional activity of SMRT (silencing mediator of retinoid and thyroid hormone receptor) by dissociation of TR and SMRT (7). Another example is that thyroid hormone promotes cell proliferation via nongenomic actions in the chick chorioallantoic membrane model (4) and in glioma cells (5). The nongenomic actions of thyroid hormone include generation of second messengers directly involved in signaling pathways that include the phosphatidylinositol 3-kinase (PI3K) (2, 12, 15, 16) or mitogen-activated protein kinase (MAPK) (11, 13, 17, 20) pathways. In a number of studies, T3 acted by stimulating the PI3K pathway, although this has not been demonstrated for T4. In human skin fibroblasts, Cao et al. (2) elucidated a T3-dependent signaling cascade leading to ZAKI-4α expression via mammalian target of rapamycin (mTOR) activation. The mTOR activation was mediated by a PI3K-Akt/PKB signaling cascade, because T3 induced phosphorylation of Akt/PKB more rapidly than that of mTOR. The T3-dependent phosphorylations were blocked both by PI3K inhibitors and by expression of a dominant-negative PI3K. The regulation of PI3K pathway by T3 was altered in gastric cancer, raising the possibility that changes in nongenomic signaling by T3 play a potential role in disease states (15). Lei and colleagues (12) demonstrated that T3 stimulated the PI3K/PKB pathway via the Src family of tyrosine kinases. In this system, activation of both Src kinase and PI3K was required for the T3-induced stimulation of Na-K-ATPase activity and its cell surface expression in adult rat alveolar epithelial cells (12). Both T4 and T3 thyroid hormones nongenomically regulate signal transduction pathways other than the PI3K pathway, including MAP kinases. For example, T3 activates MAPK/ERK1/2 in alveolar epithelial cells, stimulating the sodium pump in a dose- and time-dependent manner (11). In prior work, Lin et al. (13) showed that thyroid hormone-enhanced IFN-γ induced antiviral activity in HeLa cells, which lack thyroid hormone receptors. This effect was activated by T4, T4-agarose, and, to a lesser extent, T3. This effect also required activation of the MAPK cascade and an interaction with the STAT1α pathway that is activated by the IFN-γ (13). Proangiogenic effects of thyroid hormone and its analogs also depend on ERK1/2 signaling in a chick chorioallantoic membrane model (17). T4, T4-agarose, and the thyroid hormone analog 3,5-diiodothyropropionic acid (DITPA) stimulated angiogenesis in this model, and the magnitude of the angiogenic effect was similar to that of VEGF and basic FGF. Either tetraiodothyroacetic acid (tetrac), a known inhibitor of binding of T4 to plasma membrane integrins, or a MAPK pathway inhibitor inhibited DITPA-induced angiogenesis. In human osteoblast-like cells, both T3 and T4 activated ERK, which resulted in DNA synthesis and cell proliferation (20). Thus there is accumulating evidence that thyroid hormones and their analogs can have rapid nongenomic effects and stimulate more than one signal transduction pathway. It is uncertain whether there is cross talk between different pathways that are stimulated by thyroid hormones. T4 and T3 are able to activate other intracellular signal transduction cascades beyond PI3K and MAPK. Acting independently of TR, thyroid hormone modulates the activity of the plasma membrane Na+/H+ exchanger (10), Ca2+-dependent stimulation of adenosine triphosphatase (23), and other ion pumps or channels [inward potassium channel (19) and sodium current (9) in cardiac myocytes]. They also stimulate the guanosine triphosphatase activity of synaptosomes (8). Studies of thyroid hormone action on cell surface events, such as calcium efflux (3, 18) or glucose uptake (21, 22), several decades ago implied the existence of one or more plasma membrane receptors for T3 or T4. Recently, integrin-αvβ3 has been reported as a cell surface receptor for T4 in CV-1 cells, a monkey fibroblast cell line that lacks functional thyroid hormone receptors (1). Inhibition of the proangiogenic effects of thyroid hormone in chick chorioallantoic membrane model by LM609, a monoclonal antibody directed against αvβ3-integrin, suggests the involvement of αvβ3 as a surface receptor (4). Lin et al. (14) studied the role of l-thyroxine and 3,5,3′ triiodo-l-thyronine in cell proliferation of human glioma cells and the contributions of MAPK (ERK1/2) and PI3K pathways in the actions of T3 and T4 (Fig. 1). They demonstrate that T3 and T4 activate ERK1/2 and proliferating cell nuclear antigen (PCNA) accumulation in a concentration-dependent manner. Although ERK activation occurred within 30 min, PCNA accumulation was seen at 24 h. In contrast, activation of PI3K with phosphorylation of its p85 subunit occurred in the U-87 MG cells treated with T3, but not with T4. The T3-induced activation of PI3K was blocked by Arg-Gly-Asp, indicating a role of integrin receptors. While the activation of the ERK1/2 pathway was necessary for thyroid hormone-induced cell proliferation in these glioma cells, the T3-induced PI3K activation caused nuclear accumulation of TRα, but not TRβ1. PI3K activation by T3 was required for T3-induced expression of hypoxia-inducible factor-1α. Taken in combination, their results suggest that there are two different receptor sites for thyroid hormones on one integrin molecule that cause downstream activation of ERK and/or PI3K. T3 binds to both sites and activates both the ERK and PI3K pathways, whereas T4 only activates ERK1/2 after binding to only one of the two surface integrin sites. Fig. 1. Specificity of thyroid hormone signaling through plasma membrane integrins. The plasma membrane αvβ3-integrin has distinct binding sites for 3,5,3′-triiodo-l-thyronine (T3) and l-thyroxine (T4). One binding site binds only T3 and ... This work reveals the novel finding of specificity of T3 and T4 acting on a single cell surface receptor at apparently distinct sites within the molecule that regulate activation of separate downstream signaling pathways. Like the glioma cells, alveolar epithelial cells also demonstrate T3 activation of both the MAPK and PI3K pathways, but the ligand to which T3 binds in that system has not been determined yet. It will be interesting to determine whether a single integrin molecule can differentially activate nongenomic signaling pathways depending on the specific ligands. The studies of Lin et al. (13, 14) add significantly to our knowledge of the rapid nongenomic actions of thyroid hormones, demonstrating a sophisticated specificity exerted at the cell surface binding of hormone to plasma membrane integrin that affects TR activity and signaling pathway activation. Another unresolved question is whether there are intracellular interactions between the PI3K and MAPK signaling pathways after they are triggered by thyroid hormones at the plasma membrane. These findings are important biologically and also offer the opportunity to target specific pathways that may be manipulated in treating pathological states.

Comparative Analysis of Pain Behaviours in Humanized Mouse Models of Sickle Cell Anemia.

Pain is a hallmark feature of sickle cell anemia (SCA) but management of chronic as well as acute pain remains a major challenge. Mouse models of SCA are essential to examine the mechanisms of pain and develop novel therapeutics. To facilitate this effort, we compared humanized homozygous BERK and Townes sickle mice for the effect of gender and age on pain behaviors. Similar to previously characterized BERK sickle mice, Townes sickle mice show more mechanical, thermal, and deep tissue hyperalgesia with increasing age. Female Townes sickle mice demonstrate more hyperalgesia compared to males similar to that reported for BERK mice and patients with SCA. Mechanical, thermal and deep tissue hyperalgesia increased further after hypoxia/reoxygenation (H/R) treatment in Townes sickle mice. Together, these data show BERK sickle mice exhibit a significantly greater degree of hyperalgesia for all behavioral measures as compared to gender- and age-matched Townes sickle mice. However, the genetically distinct “knock-in” strategy of human α and β transgene insertion in Townes mice as compared to BERK mice, may provide relative advantage for further genetic manipulations to examine specific mechanisms of pain.

Electroacupuncture in conscious free-moving mice reduces pain by ameliorating peripheral and central nociceptive mechanisms.

Integrative approaches such as electroacupuncture, devoid of drug effects are gaining prominence for treating pain. Understanding the mechanisms of electroacupuncture induced analgesia would benefit chronic pain conditions such as sickle cell disease (SCD), for which patients may require opioid analgesics throughout life. Mouse models are instructive in developing a mechanistic understanding of pain, but the anesthesia/restraint required to administer electroacupuncture may alter the underlying mechanisms. To overcome these limitations, we developed a method to perform electroacupuncture in conscious, freely moving, unrestrained mice. Using this technique we demonstrate a significant analgesic effect in transgenic mouse models of SCD and cancer as well as complete Freund’s adjuvant-induced pain. We demonstrate a comprehensive antinociceptive effect on mechanical, cold and deep tissue hyperalagesia in both genders. Interestingly, individual mice showed a variable response to electroacupuncture, categorized into high-, moderate-, and non-responders. Mechanistically, electroacupuncture significantly ameliorated inflammatory and nociceptive mediators both peripherally and centrally in sickle mice correlative to the antinociceptive response. Application of sub-optimal doses of morphine in electroacupuncture-treated moderate-responders produced equivalent antinociception as obtained in high-responders. Electroacupuncture in conscious freely moving mice offers an effective approach to develop a mechanism-based understanding of analgesia devoid of the influence of anesthetics or restraints.

Genetic inactivation of calpain-1 attenuates pain sensitivity in a humanized mouse model of sickle cell disease.

Calpain-1, a calcium-activated cysteine protease, is ubiquitously expressed in hematopoietic cells, and is known to play a functional role in a myriad of cellular processes by regulating limited cleavage of multiple substrates.[1][1] Using a calpain-1 null model (CKO) previously generated in our

Cell-specific signal transduction pathways regulating Na+-K+-ATPase. Focus on “Short-term effects of thyroid hormones on the Na+-K+-ATPase activity of chick embryo hepatocytes during development: focus on signal transduction”

the na+-k+-atpase is a complex integral membrane protein that carries out active transport of sodium and potassium across the cell plasma membrane, maintaining the ionic gradients. Each subunit has multiple isozymes that are expressed in tissue and developmental specific fashion. In addition to