Maneesh Bhargava

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.

Proteomic Profiles in Acute Respiratory Distress Syndrome Differentiates Survivors from Non-Survivors

Acute Respiratory Distress Syndrome (ARDS) continues to have a high mortality. Currently, there are no biomarkers that provide reliable prognostic information to guide clinical management or stratify risk among clinical trial participants. The objective of this study was to probe the bronchoalveolar lavage fluid (BALF) proteome to identify proteins that differentiate survivors from non-survivors of ARDS. Patients were divided into early-phase (1 to 7 days) and late-phase (8 to 35 days) groups based on time after initiation of mechanical ventilation for ARDS (Day 1). Isobaric tags for absolute and relative quantitation (iTRAQ) with LC MS/MS was performed on pooled BALF enriched for medium and low abundance proteins from early-phase survivors (n = 7), early-phase non-survivors (n = 8), and late-phase survivors (n = 7). Of the 724 proteins identified at a global false discovery rate of 1%, quantitative information was available for 499. In early-phase ARDS, proteins more abundant in survivors mapped to ontologies indicating a coordinated compensatory response to injury and stress. These included coagulation and fibrinolysis; immune system activation; and cation and iron homeostasis. Proteins more abundant in early-phase non-survivors participate in carbohydrate catabolism and collagen synthesis, with no activation of compensatory responses. The compensatory immune activation and ion homeostatic response seen in early-phase survivors transitioned to cell migration and actin filament based processes in late-phase survivors, revealing dynamic changes in the BALF proteome as the lung heals. Early phase proteins differentiating survivors from non-survivors are candidate biomarkers for predicting survival in ARDS.

Protein expression profile of rat type two alveolar epithelial cells during hyperoxic stress and recovery

In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized and are similar to changes in human acute respiratory distress syndrome. In the injured lung, alveolar type two (AT2) epithelial cells play a critical role in restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. We applied an unbiased systems-level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQ with tandem mass spectrometry. Of the 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene set enrichment analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A novel short time-series expression miner algorithm identified protein clusters with coherent changes during injury and repair. We concluded that coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

Mechanisms of alveolar epithelial chloride absorption.

over the past decade, the important role of active alveolar epithelial ion transport in keeping the alveolus dry and in removing alveolar edema fluid after premature birth or alveolar flooding with heart failure or acute lung injury/acute respiratory distress syndrome (ALI/ARDS) has become widely

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.

Adalimumab induced pulmonary sarcoid reaction.

Sarcoidosis is a multisystem granulomatous inflammatory disease of unknown etiology. There is evidence that Tumor Necrosis Factor alpha (TNF-α) antagonists are useful in the treatment of advanced or refractory disease. However, sarcoidosis-like reaction has been reported with TNF-α blockade in other inflammatory conditions. Here we report a case of sarcoid-like reaction in a patient with psoriatic arthritis shortly after initiation of adalimumab therapy. Stopping adalimumab and systemic anti-inflammatory therapy with corticosteroids resulted in resolution of pulmonary symptoms and chest radiographic findings. Though TNF-α plays a critical role in pathogenesis of sarcoidosis, the development of sarcoid reaction with TNF-α blockade is paradoxical and the mechanism of this response remains unknown. TNF-α induced sarcoid-reaction could involve multiple organs. Its development with one agent does not preclude therapy with other TNF-α blockers.

Chyloptysis causing plastic bronchitis

Chyloptysis is a rare clinical problem that is associated with conditions affecting lymphatic channels in the thorax. Diagnosis is usually made when the patients present with expectoration of milky-white sputum or of thick tenacious mucus in the shape of smaller bronchi (bronchial cast). Typically the symptoms resolve after coughing up of the bronchial casts. Pleural, mediastinal, pulmonary or lymphatic abnormalities result in chyloptysis. Lymphangiography and detection of lipids (cholesterol or triglycerides) in sputum help to establish the diagnosis. However, lymphangiography may not be positive in all patients. We report 2 patients with chyloptysis and bronchial casts with different etiologies. Abnormal lymphatics were demonstrated in one of our cases, but the second patients lymphangiogram was normal. In this patient we suspect that high venous filling pressures due to congestive heart failure had a causative effect in the setting of compromised lymphatic drainage in the thorax due to a prior history of radiation therapy to the chest for lymphoma.

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

Traveling Thrombus in the Right Atrium: Is It the Final Destination?

Right heart thrombus is rare in structurally normal heart. Here, we report a 74-year-old man with a right atrial thrombus who presented with shortness of breath.