Biomedical subjects

Shahid Umar

Publications and source records attributed to Shahid Umar.

5 recordsLinked to original sources

PubMed2006

Dual alterations in casein kinase I-epsilon and GSK-3beta modulate beta-catenin stability in hyperproliferating colonic epithelia.

Casein kinase I (CKI)-epsilon and GSK-3beta phosphorylate beta-catenin at Ser(45) (beta-cat(45)) and Thr(41)/Ser(37,33) (beta-cat(33,37,41)) residues, thereby facilitating its ubiquitination and proteasomal degradation. We used a Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model to determine Ser/Thr phosphorylation and biological function of beta-catenin during crypt hyperproliferation. TMCH was associated with 3-fold and 3.3-fold increases in CKI-epsilon cellular abundance and 2-fold and 1.8-fold increase in its activity at 6 and 12 days after infection, respectively. beta-Catenin coimmunoprecipitated with both cellular and nuclear CKI-epsilon and cellular axin at these time points. Cellular beta-catenin was constitutively phosphorylated at Ser(45) and underwent subcellular redistribution to cytoskeletal and nuclear fractions at days 6 and 12 of TMCH, respectively. beta-cat(33,37,41), however, exhibited only subtle changes in either phosphorylation status or subcellular distribution even after blocking proteasomal degradation in vivo. Interestingly, GSK-3beta underwent increased phosphorylation at Ser(9), leading to 40% and 70% decreases in its activity at these time points, respectively. Coimmunoprecipitation studies exhibited strong association of GSK-3beta with PKC-zeta at either time point. Cellular beta-cat(45) stabilized and, along with unphosphorylated beta-catenin, underwent nuclear translocation, associated with nuclear accumulated Tcf-4 and cAMP response element binding protein binding protein, and was significantly acetylated, leading to increases in DNA binding. Priming of beta-catenin at Ser(45) exists in vivo. However, beta-cat(45) does not necessarily enter the degradation pathway. Impairment in linking beta-cat(45) to subsequent GSK-3beta-mediated phosphorylation and degradation may account for increased steady-state levels of both unphosphorylated as well as Ser(45)-phosphorylated beta-catenin, which may be causally linked to increases in cell census during TMCH.

Animals↗

PubMed2006

Citrobacter rodentium-induced NF-kappaB activation in hyperproliferating colonic epithelia: role of p65 (Ser536) phosphorylation.

1. The transcription factors of the NF-kappaB/Rel family form dimeric complexes that control expression of various genes involved in inflammation and proliferation. 2. During transmissible murine colonic hyperplasia (TMCH) induced by Citrobacter rodentium, nuclear translocation of NF-kappaB in isolated colonic crypts increased 3 day's post-infection and continued over 12 days paralleling peak hyperplasia. Antibody supershifts for both p65/p50 hetero- and p50/p50 homodimers occurred. Expression levels of both p50 and p65 subunits increased in cytosolic/nuclear extracts and correlated with NF-kappaB activation kinetics. IkappaB alpha levels decreased during this time. 3. Phosphorylation of IKK alpha (at Ser(176/180)) and -beta (at Ser(177/181)) increased significantly during TMCH suggesting activation in vivo. 4. p65-Ser536 (p65(536)) exhibited increased phosphorylation on immunoblotting and immunohistochemistry (IHC) both at day 6 and 12 TMCH. p65(536) translocated to nucleus and interacted with transcriptional coactivator CREB binding protein (CBP). 5. Proteasomal inhibitor bortezomib (Velcade) caused accumulation of Ser(32/36)-phosphorylated IkappaB alpha and significant inhibition of NF-kappaB activity in vivo. Velcade also blocked nuclear translocation of activated p65: both immunoblotting and IHC failed to detect p65(536) nuclear immunoreactivity. Velcade, however, did not abrogate TMCH. 6. p65 interacted strongly with ribosomal S6 kinase 1 (RSK-1) during coimmunoprecipitation but not with IKK alpha or -beta. 7. Thus, NF-kappaB activation during TMCH involves both IkappaB alpha degradation and p65-Ser536 phosphorylation. p65/RSK-1 interaction and concomitant increase in p65(536) complexed with CBP may be important in modulating NF-kappaB activity in vivo. Activated NF-kappaB, besides modulating proliferation, may aid in providing protective immunity against C. rodentium infection in vivo.

Animals↗

PubMed2006

Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation.

We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl- current (ISC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane ISC generation. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC alpha-, beta1-, gamma-, and epsilon-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-alpha membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-beta1, decreased PKC-beta2 membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta1 and a significant reduction in PKC-epsilon mass occurred. Thus activated PKC-beta1 and/or PKC-epsilon prevented, whereas activated PKC-alpha facilitated, apical membrane ISC-cAMP. PKC-beta-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC beta-isoform.

Colon↗

PubMed2006

Chronic PKC-beta2 activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting.

We investigated the effects of chronically applied PKC-stimulating phorbol esters on subcellular CFTR expression and localization in polarized HT-29 Cl.19A monolayers. Modulation of PKC activity with the PKC-beta-specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or nonisoform-selective PMA altered monolayer CFTR immunofluorescence. A decrease in the CFTR signal within the luminal cellular pole was noted with both phorbol esters. Volumetric analysis of the intracellular CFTR signal revealed that both compounds promoted CFTR accumulation into punctate vesicle-like structures found adjacent to the cellular tight junction [labeled with zona occludens (ZO)-1 antibody], extending basally (DOPPA) into the cell. Puncta were more frequent with DOPPA and larger in size with PMA. DOPPA also promoted ZO-1 accumulation at tricellular corners associated with enhanced CFTR puncta number. The observed loss of CFTR immunofluorescence signal induced by low-dose PMA was related to CFTR sequestration into fewer cytoplasmic puncta and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters downregulated steady-state cellular CFTR mRNA levels by 70%. However, the effects of DOPPA and PMA were largely independent of CFTR biosynthesis: expression levels were 80-85% of control, and the glycosylation status of immunoprecipitated protein remained largely unchanged. Thus changes in cellular CFTR localization correlated with our companion study showing that PMA-induced inhibition of transcellular cAMP-dependent short-circuit current (ISC) was accompanied by cytoplasmic PKC-beta2 accumulation and modest activation of PKC-beta1 and PKC-epsilon. The inhibitory effect of DOPPA on ISC was related solely to increased cytoplasmic PKC-beta2 levels. Thus PKC-beta2 is hypothesized to participate in the regulation of CFTR apical plasma membrane targeting within the constitutive cellular biosynthetic pathway.

Colon↗

PubMed2005

Epithelial proliferation induces novel changes in APC expression.

The role of wild-type adenomatous polyposis coli (APC) protein in native epithelia is poorly understood. The present study examined the relationships between wild-type APC and beta-catenin expression in an established model of hyperproliferation, transmissible murine colonic hyperplasia (TMCH). Distal colonic crypts isolated from normal or TMCH mice were: (i) fractionated into cytosolic and nuclear components for Western blotting and immunoprecipitation (IP), (ii) extracted for total RNA isolation for Northern blotting and, (iii) analysed immunohistochemically by confocal microscopy. Western blots performed sequentially through day 12 TMCH with N-terminal APC antibodies revealed increased abundance of approximately 312 kDa (p312) protein by day 6 (4.0 +/- 0.75-fold, n = 6) that peaked by day 9, before declining by day 12. A approximately 130 kDa (p130) band appeared at day 9 and increased by day 12 (1.5 +/- 0.11-fold, n = 6). A C-terminal antibody detected only p312. APC mRNA level did not change during TMCH and appearance of p130 was not due to alternative splicing. Co-IP with N-terminal anti-APC antibodies, revealed APC's association with beta-catenin both at day 6 and day 12. p130, but not p312, associated predominantly with beta-catenin at day 12 during co-IP with anti-beta-catenin. p130 also selectively accumulated in the nucleus, bound to nuclear beta-catenin at day 12. Immunocytochemistry with N-terminal antibodies revealed an increasing crypt base : surface gradient of APC within the apical pole/apical-lateral membranes at day 6. At day 12, intense apical/cytoplasmic and occasional nuclear staining along the longitudinal crypt axis was observed. Full-length APC increases during epithelial hyperproliferation and may represent a homoeostatic response. The dramatic increase in cytoplasmic and sporadic nuclear APC staining at day 12 with N-terminal antibodies may represent p130. The nuclear accumulation of p130 may be a novel mechanism regulating nuclear beta-catenin function during TMCH.

Animals↗