Jagadeesh Janjanam

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk‐secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI‐TOF/TOF MS and 1D‐Gel‐LC‐MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT‐PCR and Western blotting. The 1D‐Gel‐LC‐MS/MS and 2DE‐MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC‐specific proteins that will help the researchers to understand the molecular events taking place during lactation.

PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration

Phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in monocyte chemotactic protein 1–induced cytoskeleton remodeling, facilitating human aortic smooth muscle cell migration.

DIGE based proteome analysis of mammary gland tissue in water buffalo (Bubalus bubalis): Lactating vis-a-vis heifer

UNLABELLED Mammary gland is an exocrine and sebaceous gland made up of branching network of ducts that end in alveoli. Milk is synthesized in the alveoli and secreted into alveolar lumen. Mammary gland represents an ideal system for the study of organogenesis that undergoes successive cycles of pregnancy, lactation and involution. To gain insights on the molecular events that take place in pubertal and lactating mammary gland, we have identified 43 differentially expressed proteins in mammary tissue of heifer (non-lactating representing a virgin mammary gland), and lactating buffaloes (Bubalus bubalis) by 2D-difference gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty one proteins were upregulated during lactation whereas 8 proteins were upregulated in heifer mammary gland significantly (p<0.05). Bioinformatics analyses of the identified proteins showed that a majority of the proteins are involved in metabolic processes. The differentially expressed proteins were validated by real-time PCR and Western blotting. We observed differential expressions of certain new proteins including EEF1D, HSPA5, HSPD1 and PRDX6 during lactation which have not been reported before. The differentially expressed proteins were mapped to available biological pathways and networks involved in lactation. This study signifies the importance of some proteins which are preferentially expressed during lactation and in heifer mammary gland. BIOLOGICAL SIGNIFICANCE This work is important because we have generated information in water buffalo (B. bubalis) for the first time which is the major milk producing animal in Indian Subcontinent. Out of a present production of 133milliontons of milk produced in India, contribution of buffalo milk is around 54%. Its physiology is somewhat different from the lactating cows. Buffalo milk composition varies from cow milk in terms of higher fat and total solid content, which confers an advantage in preparation of specialized cheese, curd and other dairy products. Being a major milk producing animal in India it is highly essential to understand the lactation associated proteins in the mammary gland of buffalo. In the present investigation our attempt has been to identify new protein evidences which are expressed in lactating buffalo mammary gland and have not been reported before. The findings reported in the present study will help in understanding the lactation biology of buffalo mammary gland in particular and the mammary gland biology in general.

P115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine-induced vascular smooth muscle cell migration and proliferation

Although the involvement of Rho proteins in the pathogenesis of vascular diseases is well studied, little is known about the role of their upstream regulators, the Rho guanine nucleotide exchange factors (RhoGEFs). Here, we sought to identify the RhoGEFs involved in monocyte chemotactic protein 1 (MCP1)–induced vascular wall remodeling. We found that, among the RhoGEFs tested, MCP1 induced tyrosine phosphorylation of p115 RhoGEF but not of PDZ RhoGEF or leukemia-associated RhoGEF in human aortic smooth muscle cells (HASMCs). Moreover, p115 RhoGEF inhibition suppressed MCP1-induced HASMC migration and proliferation. Consistent with these observations, balloon injury (BI) induced p115 RhoGEF tyrosine phosphorylation in rat common carotid arteries, and siRNA-mediated down-regulation of its levels substantially attenuated BI-induced smooth muscle cell migration and proliferation, resulting in reduced neointima formation. Furthermore, depletion of p115 RhoGEF levels also abrogated MCP1- or BI-induced Rac1–NFATc1–cyclin D1–CDK6–PKN1–CDK4–PAK1 signaling, which, as we reported previously, is involved in vascular wall remodeling. Our findings also show that protein kinase N1 (PKN1) downstream of Rac1–cyclin D1/CDK6 and upstream of CDK4–PAK1 in the p115 RhoGEF–Rac1–NFATc1–cyclin D1–CDK6–PKN1–CDK4–PAK1 signaling axis is involved in the modulation of vascular wall remodeling. Of note, we also observed that CCR2–Gi/o–Fyn signaling mediates MCP1-induced p115 RhoGEF and Rac1 GTPase activation. These findings suggest that p115 RhoGEF is critical for MCP1-induced HASMC migration and proliferation in vitro and for injury-induced neointima formation in vivo by modulating Rac1–NFATc1–cyclin D1–CDK6–PKN1–CDK4–PAK1 signaling.

Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis

MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented.

Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1

Monocyte chemotactic protein 1 (MCP1) stimulates phosphorylation of cortactin on Y421 and Y446 residues in a time-dependent manner and phosphorylation at Y446 but not Y421 residue is required for MCP1-induced CDK-interacting protein 1 (p21Cip1) nuclear export and degradation in facilitating human aortic smooth muscle cell (HASMC) proliferation. In addition, MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation are dependent on Fyn activation. Upstream to Fyn, MCP1 stimulated C-C chemokine receptor type 2 (CCR2) and Gi/o and inhibition of either one of these molecules using their specific antagonists or inhibitors attenuated MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Cortactin phosphorylation at Y446 residue is also required for another G protein-coupled receptor (GPCR) agonist, thrombin-induced p21Cip1 nuclear export and its degradation in promoting HASMC proliferation. Quite interestingly, the receptor tyrosine kinase (RTK) agonist, platelet-derived growth factor-BB (PDGF-BB)-induced p21Cip1 degradation and HASMC proliferation do not require cortactin tyrosine phosphorylation. Together, these findings demonstrate that tyrosine phosphorylation of cortactin at Y446 residue is selective for only GPCR but not RTK agonist-induced nuclear export and proteolytic degradation of p21Cip1 in HASMC proliferation.

LIM and cysteine-rich domains 1 is required for thrombin-induced smooth muscle cell proliferation and promotes atherogenesis

Restenosis arises after vascular injury and is characterized by arterial wall thickening and decreased arterial lumen space. Vascular injury induces the production of thrombin, which in addition to its role in blood clotting acts as a mitogenic and chemotactic factor. In exploring the molecular mechanisms underlying restenosis, here we identified LMCD1 (LIM and cysteine-rich domains 1) as a gene highly responsive to thrombin in human aortic smooth muscle cells (HASMCs). Of note, LMCD1 depletion inhibited proliferation of human but not murine vascular smooth muscle cells. We also found that by physically interacting with E2F transcription factor 1, LMCD1 mediates thrombin-induced expression of the CDC6 (cell division cycle 6) gene in the stimulation of HASMC proliferation. Thrombin-induced LMCD1 and CDC6 expression exhibited a requirement for protease-activated receptor 1-mediated Gαq/11-dependent activation of phospholipase C β3. Moreover, the expression of LMCD1 was highly induced in smooth muscle cells located at human atherosclerotic lesions and correlated with CDC6 expression and that of the proliferation marker Ki67. Furthermore, the LMCD1- and SMCαactin-positive cells had higher cholesterol levels in the atherosclerotic lesions. In conclusion, these findings indicate that by acting as a co-activator with E2F transcription factor 1 in CDC6 expression, LMCD1 stimulates HASMC proliferation and thereby promotes human atherogenesis, suggesting an involvement of LMCD1 in restenosis.

Role of extracellular matrix in breast cancer development: a brief update

Evidence is increasing on the crucial role of the extracellular matrix (ECM) in breast cancer progression, invasion and metastasis with almost all mortality cases owing to metastasis. The epithelial-mesenchymal transition is the first signal of metastasis involving different transcription factors such as Snail, TWIST, and ZEB1. ECM remodeling is a major event promoting cancer invasion and metastasis; where matrix metalloproteinases (MMPs) such as MMP-2, -9, -11, and -14 play vital roles degrading the matrix proteins for cancer spread. The β-D mannuronic acid (MMP inhibitor) has anti-metastatic properties through inhibition of MMP-2, and -9 and could be a potential therapeutic agent. Besides the MMPs, the enzymes such as LOXL2, LOXL4, procollagen lysyl hydroxylase-2, and heparanase also regulate breast cancer progression. The important ECM proteins like integrins (b1-, b5-, and b6- integrins), ECM1 protein, and Hic-5 protein are also actively involved in breast cancer development. The stromal cells such as tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and adipocytes also contribute in tumor development through different processes. The TAMs become proangiogenic through secretion of VEGF-A and building vessel network for nourishment and invasion of the tumor mass. The latest developments of ECM involvement in breast cancer progression has been discussed in this review and this study will help researchers in designing future work on breast cancer pathogenesis and developing therapy targeted to the ECM components.

A new role for cofilin in retinal neovascularization.

ABSTRACT Pak1 plays an important role in several cellular processes, including cell migration, but its role in pathological angiogenesis is not known. Here, we have determined its role in pathological retinal angiogenesis using an oxygen-induced retinopathy (OIR) model. VEGFA induced phosphorylation of Pak1 and its effector cofilin in a manner that was dependent on time as well as p38MAPKβ (also known as MAPK11) in human retinal microvascular endothelial cells (HRMVECs). Depletion of the levels of any of these molecules inhibited VEGFA-induced HRMVEC F-actin stress fiber formation, migration, proliferation, sprouting and tube formation. In accordance with these observations, hypoxia induced Pak1 and cofilin phosphorylation with p38MAPKβ being downstream to Pak1 and upstream to cofilin in mouse retina. Furthermore, Pak1 deficiency abolished hypoxia-induced p38MAPKβ and cofilin phosphorylation and abrogated retinal endothelial cell proliferation, tip cell formation and neovascularization. In addition, small interfering RNA (siRNA)-mediated downregulation of p38MAPKβ or cofilin levels in the wild-type mouse retina also diminished endothelial cell proliferation, tip cell formation and neovascularization. Taken together, these observations suggest that, although the p38MAPKβ–Pak1–cofilin axis is required for HRMVEC migration, proliferation, sprouting and tubulogenesis, Pak1–p38MAPKβ–cofilin signaling is also essential for hypoxia-induced mouse retinal endothelial cell proliferation, tip cell formation and neovascularization. Summary: Pak1 and its downstream effector cofilin play an essential role in VEGFA-induced angiogenic events in human retinal microvascular endothelial cells in vitro and hypoxia-induced retinal neovascularization in vivo.